Total synthesis and conformational analysis of apratoxin C

Article abstract of DOI:10.1021/jo501130b

Total synthesis of apratoxin C, a cyanobacterial cyclodepsipeptide with highly potent cytotoxicity against some cancer cell lines, was achieved using the apratoxin A synthetic strategy developed by us. To elucidate the relationship between conformation and activity, the tertiary structure of apratoxin C was analyzed by NMR spectroscopy. We obtained 37 ROEs and five ³J_H,H values, which were translated into distance and dihedral angle constraints, respectively. Molecular modeling was performed with a restrained conformational search by a distance geometry method. The lowest energy structure indicated that the methyl group at C37 and the isopropyl group at C39 play critical roles in maintaining the conformation, whereas the methyl group at C34 does not. Moreover, we confirmed that apratoxin A and C possess similar conformations, providing a likely explanation for their nearly equivalent cytotoxicities.

Full text of DOI:10.1021/jo501130b

Article
pubs.acs.org/joc
Total Synthesis and Conformational Analysis of Apratoxin C
Yuichi Masuda,† Jun Suzuki,† Yuichi Onda,†,‡ Yuta Fujino,† Masahito Yoshida,† and Takayuki Doi*,†
†Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aza-Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan
‡Mitsubishi Tanabe Pharma Corporation, 2-2-50, Kawagishi, Toda-shi, Saitama 335-8505, Japan
S
* Supporting Information
ABSTRACT: Total synthesis of apratoxin C, a cyanobacterial
cyclodepsipeptide with highly potent cytotoxicity against some
cancer cell lines, was achieved using the apratoxin A synthetic
strategy developed by us. To elucidate the relationship
between conformation and activity, the tertiary structure of
apratoxin C was analyzed by NMR spectroscopy. We obtained
3
37 ROEs and five JH,H values, which were translated into
distance and dihedral angle constraints, respectively. Molecular
modeling was performed with a restrained conformational
search by a distance geometry method. The lowest energy
structure indicated that the methyl group at C37 and the
isopropyl group at C39 play critical roles in maintaining the
conformation, whereas the methyl group at C34 does not.
Moreover, we confirmed that apratoxin A and C possess similar conformations, providing a likely explanation for their nearly
equivalent cytotoxicities.
addition, we reported the synthesis and biological evaluation of
apratoxin analogues in which one of the residues was replaced
with an azido-derivatized amino acid.15 Recently, Luesch et al.
conducted a structure-activity relationship (SAR) study of
apratoxins and developed an apratoxin A/E hybrid analogue
with improved in vivo antitumor activity.21,22 According to the
above-mentioned studies, the modification of apratoxins could
affect not only the local structure but also the main chain
conformation because of the flexibility of the molecular frame
of cyclic peptides. Luesch et al. analyzed the tertiary structures
of apratoxin A (1), apratoxin B (2), and E-dehydroapratoxin A
(5) by NMR spectroscopy.4,5 They indicated that the difference
in the cytotoxicities could be derived from conformational
changes; thus, it is essential to understand the effect of
modification on the conformations for SAR and drug design
based on the cyclodepsipeptide scaffolds. Since the tert-butyl
group in apratoxin A (1) is thought to be important to regulate
its conformation, we focused on apratoxin C (3), which
possesses an isopropyl group instead of the tert-butyl group in
1. Herein, we report the synthesis of apratoxin C (3) and the
analysis of the conformation by NMR spectroscopy.
INTRODUCTION
■
Marine cyanobacteria produce a number of secondary
metabolites that possess interesting molecular architectures
and biological properties.1−3 Among the metabolites, cyano-
bacterial cyclodepsipeptides, which have unique scaffolds and
nonribosomally synthesized peptide motifs, attract considerable
attention as novel pharmaceuticals because of their striking
biological activities.1−3 One example is the apratoxin family
(Figure 1), which exhibits highly potent cytotoxicity against
some cancer cell lines.4−9 Apratoxins A−D (1−4)4−6 are the
cyclodepsipeptides that feature a proline residue, N-methylated
amino acids, a modified cysteine residue, and a dihydroxylated
fatty acid moiety (Figure 1). Because of their unique structure
and biological activity, several researchers have demonstrated
the total syntheses of apratoxins; Forsyth’s group was the first
to complete the total synthesis of apratoxin A (1).10,11
Furthermore, two groups including us have achieved total
synthesis of 1.12−14 We have also demonstrated the solid-phase
total synthesis of apratoxin A (1) and its analogues.15 Recently,
the total synthesis of apratoxin D (4) was reported.16 Because
apratoxins have potential as novel anticancer lead compounds,
their mode of action has been studied by genomics and
chemical biological approaches.17−20
RESULTS AND DISCUSSION
■
The cytotoxicity of apratoxins is sensitive to certain structural
modifications in the fatty acid region. Ma et al. reported that
reversal of the configuration at C37 from 37S to 37R or
removal of the methyl group at C37 abolished the cytotoxicity
of apratoxins in oxazoline analogues.14 Our group reported that
the protection of the hydroxyl group at C35 with a triethylsilyl
(TES) group led to lack of cytotoxicity, whereas the (34R)-
diastereomer of apratoxin A showed equipotent activity.13,15 In
Total Synthesis of Apratoxin C (3). According to our
total synthesis of apratoxin A (1),12,13 synthesis of apratoxin C
(3) can be performed using a similar strategy (Scheme 1). In
principle, apratoxin C (3) can be synthesized by macro-
Received: May 22, 2014
© XXXX American Chemical Society
A
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Figure 1. Chemical structures of apratoxins.
Scheme 1. Retrosynthetic Analysis of Apratoxin C (3)
lactamization of the linear compound 6, which can be obtained
by coupling carboxylic acid 7 with tripeptide 8. Acid 7 can be
prepared from the coupling of Pro-Dtrina (3,7-dihydroxy-2,5,8-
trimethylnonanoic acid) moiety 9 with a derivative of
compound 10, followed by construction of a thiazoline ring.
Constructions of the stereogenic centers at the C34, C35, C37,
and C39 positions are vital for the synthesis of Pro-Dtrina 9,
which can be synthesized from a derivative of compound 11 via
diastereoselective crotylation23,24 of aldehyde 13 with E-crotyl
borate (E)-12. The stereogenic center at C37 can be
introduced by asymmetric hydrogenation of allylic alcohol
(E)-14 in the presence of a Ru-(S)-BINAP catalyst.25
Compound (E)-14 can be transformed from compound 15,
B
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Scheme 2. Synthesis of Pro-Dtrina 9
which is obtained by proline-catalyzed enantioselective aldol
reaction26 of acetone with isobutylaldehyde.
of the diastereomers for the next reaction. The alcohol 11 and
its diastereomer were protected with a 2,2,2-trichloroethox-
ycarbonyl (Troc) group to provide 19 and its diastereomer.
Removal of the PMB group with 2,3-dichloro-5,6-dicyano-p-
benzoquinone (DDQ), followed by coupling with Fmoc-Pro-
OH by the Yamaguchi method,30 gave compound 20 without
epimerization at the α-position of the proline moiety. The
diastereomer of 20 was separated by silica gel column
chromatography. Finally, oxidative cleavage of the terminal
alkene and subsequent oxidation of the resulting aldehyde
furnished the desired Pro-Dtrina 9.
After obtaining the desired 9, we synthesized the linear
peptide 23 leading to apratoxin C (3) (Scheme 3). After
removal of the N-Boc group in 10 (TMSOTf/2,6-lutidine, rt, 7
h),12,13 coupling of the resulting amine with 9 using N-(3-
(dimethylamino)propyl)-N′-ethylcarbodiimide (EDCI)/ 1-hy-
droxy-7-azabenzotriazole (HOAt) provided the amide 21 in a
55% yield. Thiazoline formation was performed by treatment
with trifluomethanesulfonic anhydride/triphenylphosphine
oxide (Tf2O/Ph3PO, 0 °C, 1 h),31 and subsequent removal
of the Troc group (Zn/NH4OAc, rt, 1.5 h) afforded thiazoline
22 in a 90% yield for two steps. Removal of the allyl group and
coupling of the resulting acid with tripeptide 812,13 provided the
desired 23 in a 95% yield for two steps. Finally, the removal of
the protecting groups at both the N- and C-terminus, followed
First, we investigated the synthesis of Pro-Dtrina 9 (Scheme
2). After a proline-catalyzed aldol reaction of acetone with
isobutylaldehyde,26 protection of the resulting hydroxyl group
of 16 with a p-methoxybenzyl (PMB) ether using PMB
trichloroacetimidate in the presence of trifluoromethanesul-
fonic acid provided compound 15, which was converted into
the alcohol 17 by 1,2-addition of a vinyl Grignard reagent to a
ketone moiety. Rhenium-catalyzed isomerization of allylic
alcohol with N,O-bis(trimethylsilyl) acetamide (BSA)27,28
provided 14 at an E:Z isomer ratio of 1:1, and these
diastereomers were separated by silica gel column chromatog-
raphy to afford (E)-14 and (Z)-1429 in a combined yield of
80%. Ru(OAc)2[(S)-binap]-catalyzed asymmetric hydrogena-
tion25 of (E)-14 under 90 atm of hydrogen afforded compound
1
18 in a 91% yield as a single diastereomer confirmed by H
NMR. After oxidation of the primary alcohol in 18 to the
aldehyde 13, diastereoselective crotylation of 13 with (E)-crotyl
borate (E)-12 afforded 11 and its diastereomer at a ratio of 9:1
1
(determined by H NMR) in a combined yield of 97%. The
mixture of the diastereomers was partially purified by
preparative RP-HPLC to afford pure 11, whose structure was
determined by instrumental analyses. As its selectivity was good
enough to contuinue the synthesis, we utilized 11 as a mixture
C
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Scheme 3. Total Synthesis of Apratoxin C (3)
by macrolactamization utilizing HATU32/diisopropylethyl-
amine (DIEA) under high dilution conditions (1 mM),
furnished apratoxin C (3) in a 21% yield for three steps.
Although trace amount of the diastereomers were also detected
in the reaction mixture of the macrolactamization by LC−MS,
they were readily removed by preparative HPLC. Spectroscopic
data, including optical rotation, of synthetic 3 were identical to
those of the natural product, apratoxin C (Figure S1, Table S1
in Supporting Information). Cytotoxic activities of the synthetic
apratoxin C (3) against HCT-116 cells were evaluated by a
WST assay.33,34 The cytotoxicity of apratoxin C (3) (IC50 value
4.1 nM) was found to be slightly weaker than that of apratoxin
A (1) (IC50 value 3.2 nM), which agrees with previous reports.5
Conformational Analysis of Apratoxin C (3) by NMR.
To investigate the conformation of apratoxin C (3), we
analyzed its tertiary structure by NMR. Because apratoxin C
(3) might be labile under acidic conditions in CDCl3,5 NMR
experiments were performed using CD3CN as the solvent.
Dihedral angle constraints were determined by J-coupling
constants between vicinal protons (3JH,H) using a J-based
configuration analysis (JBCA) method35 (Table S2 in
Supporting Information). For distance constraints, 2D
ROESY experiments were performed, and the cross peaks
were semiquantitatively translated into three distance constraint
categories (strong, ≤2.5 Å; medium, ≤3.5 Å; weak, ≤5.0 Å)
according to their signal intensities (Table S2 in Supporting
Information).
3JH,H values and ROEs clearly identified the major
conformation of the Dtrina region (Figure 2A), the Newman
projections of which are shown in Figure 2B−F. The carbons of
the main chain at the C33−C36, C34−C37, and C35−C38
positions were in anti orientations to avoid steric hindrance
(Figure 2B−D). On the other hand, the main chain atoms at
the C36−C39 and C37−OCO positions were in gauche
orientations (Figure 2E and F). The isopropyl group at C39
(iPr in Figure 2F) was in anti orientation against the main chain
(C37 in Figure 2F), indicating that this bulky group could be
important for maintaining the conformation. Surprisingly, the
methyl group at C37 (Me45 in Figure 2E) was also in anti
orientation to the main chain carbon (C39 in Figure 2E). This
suggests that the methyl group at C37 would play a crucial role
in maintaining the conformation. On the other hand, the
methyl group at C34 (Me44 in Figure 2B) was not in anti
orientation against the main chain (C36 in Figure 2B). The
methyl group at C34 seems not to be important for maintaining
the conformation.
Molecular modeling was performed on a MacroModel
(version 9.9) program36−38 using distance geometry followed
by a conformational search using 10,000-step Monte Carlo-
based torsional sampling with 37 distance and 5 dihedral angle
D
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not form a strong hydrogen bond with the carbonyl group of
Pro.
Because we previously accomplished the total synthesis of
apratoxin A (1),12,13 we also analyzed its tertiary structure by
NMR measured in CD3CN (Figures S3 and S4, Table S3 in
Supporting Information). The 3D structure of apratoxin A (1)
obtained was almost identical to that of apratoxin C (3) (Figure
4). The tert-butyl group is in anti orientation against the main
Figure 2. Major conformation of the Dtrina region in apratoxin C (3)
confirmed by the NMR data measured in CD3CN. (A) The structure
of the Dtrina region. (B−F) Newman projections of the Dtrina region.
3JH,H values are shown below the Newman projections and “Large” and
3
“Small” refer to the magnitude of the JH,H values, resulting in the
identification of anti or gauche orientations. Red double-headed arrows
indicate the observed ROEs (“Strong” and “Medium” represent
relative intensities), which support the proposed conformations. Blue
letters indicate the carbons of the main chain.
constraints derived from the NMR data. We applied an OPLS-
2005 force field and a generalized Born/solvent-accessible
surface area (GB/SA) solvent model.39 The calculation was
conducted in a chloroform environment.40 The stable
structures obtained are shown in Figure 3. The side chain of
Figure 4. Superposition of the lowest energy conformers of apratoxin
A (structure in red) and apratoxin C (structure in blue) obtained with
a conformational search by the distance geometry method.
chain (Figure S3F in Supporting Information) like the
isopropyl group in apratoxin C (3) (Figure 3F). This indicates
that both the tert-butyl group in apratoxin A (1) and the
isopropyl group in apratoxin C (3) play significant roles in
maintaining their conformations as bulky hydrophobic groups.
The nearly equivalent cytotoxicity of apratoxin A (1) and C (3)
could arise from the similarity of their conformations. Luesch et
al. have proposed a 3D structural model of apratoxin A (1) in
CDCl3 by NMR.4 Our structural models of apratoxin A (1) and
apratoxin C (3) in CD3CN were very similar to that of
apratoxin A (1) in CDCl3 proposed by Luesch et al.4 Apratoxin
A (1) exists in similar conformations in both CD3CN and
CDCl3.
Figure 3. Molecular modeling of apratoxin C (3) with a restrained
conformational search by the distance geometry method: (A)
superposition of top 10 stable conformers consistent with NMR
data (3JH,H and ROEs) and (B) the lowest energy conformer generated
by the conformational search.
CONCLUSION
■
To the best of our knowledge, we accomplished the total
synthesis of apratoxin C (3) for the first time. Our synthetic
strategy of apratoxin A (1)12,13 was demonstrated to be
applicable to the synthesis of apratoxin C (3) without any
major tactical alteration. On the basis of the molecular
modeling with constraints from the NMR data measured in
CD3CN, we presented the structural model of apratoxin C (3)
in an aprotic solvent. Our structural model indicated that the
methyl group at C37 and the isopropyl group at C39 play
critical roles in maintaining the conformation, whereas the
methyl group at C34 does not. In addition, we confirmed that
apratoxin A (1) and C (3) form similar conformations in
CD3CN, which is in good agreement with their comparable
cytotoxicities.
N-methylisoleucine is close to the Dtrina moiety, and this was
indicated by the ROE cross peaks between them (Table S2 in
Supporting Information). Despite apratoxin C (3) comprising a
proline and two N-methylated amino acids, all the amide bonds
were found to be in s-trans forms in this model (Figure 3).
Since the hydroxy proton at C35 is proximal to the carbonyl
oxygen of Pro in the structural model (ca. 1.8 Å in Figure 3B),
we investigated the existence of a hydrogen bond between them
by hydrogen−deuterium exchange experiment of apratoxin C in
CD3CN (Figure S2 in Supporting Information). The hydroxy
proton at C35 was exchanged to deuterium immediately upon
addition of D2O to the NMR solvent (Figure S2 in Supporting
Information), suggesting that the hydroxy group at C35 does
E
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acetate. The combined organic layers were washed with brine, dried
over MgSO4, and concentrated in vacuo. The residue was passed
through a short pad of silica gel to afford crude 17 and used for the
next reaction without further purification.
EXPERIMENTAL SECTION
■
General Techniques. All commercially available reagents were
purchased from commercial suppliers and used as received. All
solution-phase reactions were monitored by thin layer chromatography
(TLC) carried out on silica gel plates (60F-254) with UV light,
visualized by p-anisaldehyde H2SO4/ethanol solution, phosphomolyb-
dic acid/ethanol solution, or ninhydrin/acetic acid/1-butanol solution.
Flash column chromatography was performed with silica gel (40−100
μm) with the indicated solvent system. 1H NMR spectra (400 and 600
MHz) and 13C NMR spectra (100 and 150 MHz) were recorded using
To a solution of allylic alcohol 17 and Ph3SiOReO3 (219 mg, 0.431
mmol) in diethyl ether (70 mL) was added N,O-bis(trimethylsilyl)
acetamide (BSA) (4.21 mL, 17.2 mmol) at 0 °C dropwise over 30 min.
After being stirred at the same temperature for 1 h, the reaction
mixture was quenched with Et3N (1.0 mL), stirred at 0 °C for 20 min,
and concentrated in vacuo. The residue was diluted with MeOH (70
mL) and treated with K2CO3 (3.97 g, 28.7 mmol) at 0 °C for 30 min.
The reaction mixture was quenched with saturated aqueous NH4Cl,
and the aqueous layer was extracted with ethyl acetate. The combined
organic layers were washed with brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by silica gel column
chromatography (5% to 10% ethyl acetate/hexane) to afford (E)-14
(1.62 g, 5.82 mmol, 40%) and (Z)-14 (1.61 g, 5.79 mmol, 40%) as
1
the indicated solvent. Chemical shifts (δ) for H NMR spectra are
given from TMS (0.00 ppm) in CDCl3 and from residual
nondeuterated solvent peaks in other solvents (CD2Cl2, 5.32 ppm;
acetonitrile-d3, 1.94 ppm; methanol-d4, 3.30 ppm) as internal
standards. Chemical shifts (δ) for 13C NMR spectra are given from
13CDCl3 (77.0 ppm), 13CD2Cl2 (54.0 ppm), acetonitrile-d3 (118.26,
1.32 ppm), and methanol-d4 (49.0 ppm) as internal standards.
Multiplicities are reported by the following abbreviations: s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), dd (double doublet),
dt (double triplet), dq (double quartet), ddd (double double doublet),
ddt (double double triplet), dddd (double double double doublet), brs
(broad singlet), brt (broad triplet), J (coupling constants in hertz).
High-resolution mass spectra were measured on TOF-MS with EI,
FAB, or ESI probe. IR spectra were reported in reciprocal centimeters
(cm−1). Optical rotations were measured at 589 nm. Melting points
were measured on a melting point apparatus and are not corrected.
Preparative RP-HPLC was carried out by using UV detection at 214
and 254 nm.
(S)-Hydroxy-5-methylhexan-2-one (16). To a solution of D-
proline (4.50 g, 39.1 mmol) in dimethyl sulfoxide (DMSO) (520 mL)
and acetone (130 mL) was added isobutylaldehyde (9.39 g, 130
mmol) at room temperature. After being stirred at the same
temperature for 2 d, the reaction mixture was quenched with saturated
aqueous NH4Cl, and the aqueous layer was extracted with ethyl
acetate. The combined organic layers were washed with brine, dried
over MgSO4, and concentrated in vacuo. The residue was purified by
silica gel column chromatography (10% ethyl acetate/hexane) to
1
colorless oil. (E)-14: H NMR (400 MHz, CDCl3) δ 7.24 (d, J = 8.0
Hz, 2 H), 6.85 (d, J = 8.0 Hz, 2 H), 5.49 (td, J = 6.8, 0.9 Hz, 1 H), 4.43
(m, 2 H), 4.14 (d, J = 6.8 Hz, 2 H), 3.79 (s, 3 H), 3.32 (dt, J = 7.6, 4.8
Hz, 1 H), 2.24−2.14 (m, 2 H), 1.92−1.84 (m, 1 H), 0.92 (d J = 6.8
Hz, 6 H); 13C NMR (100 MHz, CDCl3) δ 159.0, 137.3, 131.1, 129.3,
125.7, 113.6, 81.8, 71.4, 59.3, 55.2, 40.9, 30.8, 18.3, 17.8, 16.7; [α]22
=
D
−0.60 (c 1.04, CHCl3); IR (neat) 3380, 2872, 1612, 1513, 1248, 1067,
1037, 821 cm−1; HRFABMS calcd for C17H27O3 [M + H]+ 279.1960,
1
found 279.1977. (Z)-14: H NMR (400 MHz, CDCl3) δ 7.23 (d, J =
8.0 Hz, 2 H), 6.85 (d, J = 8.0 Hz, 2 H), 5.71 (brt, J = 7.4 Hz, 1 H),
4.49 (d, J = 11.0 Hz, 1 H), 4.34 (d, J = 11.0 Hz, 1 H), 4.12 (dd, J =
11.3, 8.4 Hz, 1 H), 3.83 (m, 1 H), 3.79 (s, 3 H), 3.31 (ddd, J = 10.8,
2.8, 2.8 Hz, 1 H), 2.54 (dd, J = 13.2, 10.8 Hz, 1 H), 2.06−2.02 (m, 1
H), 1.86 (dd, J = 13.2, 2.4 Hz, 1 H), 1.70 (s, 3 H), 0.95 (d, J = 6.8 Hz,
6 H), 0.94 (d, J = 6.8 Hz, 6 H); 13C NMR (100 MHz, CDCl3) δ 159.2,
138.3, 130.0, 129.7, 126.8, 113.7, 80.3, 71.7, 57.9, 55.2, 32.7, 30.4, 23.5,
18.5, 16.8; [α]24 = −2.55 (c 1.15, CHCl3); IR (neat) 3419, 2960,
D
1613, 1514, 1249, 1062, 1036, 822 cm−1; HRFABMS calcd for
C17H27O3 [M + H]+ 279.1960, found 279.1951.
(3R,5S)-5-(4-Methoxybenzyloxy)-3,6-dimethylheptan-1-ol
(18). To a solution of (E)-14 (1.22 g, 4.38 mmol) in MeOH (8.7 mL)
was added Ru(OAc)2[(S)-binap] (73 mg, 0.087 mmol), and the
mixture was placed in an autoclave. The autoclave was filled with
hydrogen (90 atm) after repeated filling and purging of hydrogen (3
times). After the reaction was carried out under the appropriate
hydrogen pressure (ca. 90 atm) at 50 °C for 12 h, the reaction mixture
was diluted with MeOH and additionally stirred with florisil at room
temperature for 15 min. Then the reaction mixture was filtered
through a pad of silica gel, and the filtrate was concentrated in vacuo.
The residue was purified by silica gel column chromatography (5%
ethyl acetate/hexane) to afford 18 (1.86 g, 6.71 mmol, 91%) as a
colorless oil: 1H NMR (400 MHz, CDCl3) δ 7.27 (d, J = 8.0 Hz, 2 H),
6.87 (d, J = 8.0 Hz, 2 H), 4.49 (d, J = 10.8 Hz, 1 H), 4.39 (d, J = 10.8
Hz, 2 H), 3.80 (s, 3 H), 3.71−3.57 (m, 2 H), 3.28 (td, J = 8.0, 3.6 Hz,
1 H), 2.00−1.93 (m, 1 H), 1.74−1.68 (m, 1 H), 1.65−1.56 (m, 1 H),
1.45−1.25 (m, 3 H), 0.93−0.89 (m, 9 H); 13C NMR (100 MHz,
CDCl3) δ 158.9, 131.1, 129.3, 113.6, 81.1, 70.8, 60.8, 55.1, 39.2, 37.5,
30.0, 26.4, 20.6, 17.9, 17.5; [α]24D = −37.7 (c 1.22, CHCl3); IR (neat)
3383, 2957, 1613, 1514, 1302, 1248, 1038, 821 cm−1; HRFABMS
calcd for C17H29O3 [M + H]+ 281.2117, found 281.2109.
(3R,5S)-5-(4-Methoxybenzyloxy)-3,6-dimethylheptanal (13).
To a solution of 18 (0.756 g, 2.69 mmol) in CH2Cl2 (27 mL) were
added Et3N (1.87 mL, 13.5 mmol), DMSO (2.86 mL, 40.4 mmol),
and sulfur trioxide pyridine complex (1.07 g, 6.74 mmol) at 0 °C
under argon. After being stirred at 0 °C to room temperature for 3 h,
the mixture was quenched with H2O, and the aqueous layer was
extracted with diethyl ether. The combined organic layers were dried
over MgSO4 and concentrated in vacuo. The residue was purified by
silica gel column chromatography (0% to 5% ethyl acetate/hexane) to
afford 13 (0.691 g, 2.48 mmol, 91%) as a colorless oil: 1H NMR (400
MHz, CDCl3) δ 9.64 (t, J = 1.6 Hz, 1 H), 7.25 (d, J = 8.0 Hz, 2 H),
6.86 (d, J = 8.0 Hz, 2 H), 4.50 (d, J = 7.2 Hz, 1 H), 4.34 (d, J = 7.2 Hz,
1 H), 3.80 (s, 3 H), 3.22 (dt, J = 8.8, 4.0 Hz, 1 H), 2.34 (ddd, J = 15.6,
1
afford 16 (8.82 g, 67.7 mmol, 52%) as a colorless oil: H NMR (400
MHz, CDCl3) δ 3.81 (ddd, J = 12.4, 6.0, 3.8 Hz, 1 H), 2.91 (d, J = 3.8
Hz, 1 H), 2.62 (dd, J = 17.4, 2.8 Hz, 1 H), 2.53 (dd, J = 17.4, 9.2 Hz, 1
H), 2.20 (s, 3 H), 1.68 (m, 1 H), 0.94 (d, J = 6.8 Hz, 3 H), 0.91 (d, J =
6.8 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 210.4, 72.2, 46.9, 32.9,
30.8, 18.3, 17.7. [α]26 = −66.7 (c 1.40, CHCl3) [lit.41 [α]25 −55 (c
D
D
1.4, CHCl3)]; IR (neat) 3438, 2962, 2877, 1713, 1362, 1065 cm−1.
(S)-(4-Methoxybenzyloxy)-5-methylhexan-2-one (15). To a
solution of 16 (6.37 g, 48.9 mmol) and 4-methoxybenzyl-2,2,2-
trichloroacetimidate in tetrahydrofuran (THF) (200 mL) was added
trifluoromethanesulfonic acid (0.043 mL, 0.489 mmol) at 0 °C under
argon. After being stirred at the same temperature for 1 h, the reaction
mixture was quenched with aqueous HCl (1 M) and extracted with
ethyl acetate. The combined organic layers were washed with saturated
aqueous NaHCO3 and brine, dried over MgSO4, and concentrated in
vacuo. The residue was purified by silica gel column chromatography
(5% ethyl acetate/hexane) to afford 15 (10.8 g, 43.1 mmol, 88%) as a
1
colorless oil: H NMR (400 MHz, CDCl3) δ 3.81 (ddd, J = 12.4, 6.0,
3.8 Hz, 1 H), 2.91 (d, J = 3.8 Hz, 1 H), 2.62 (dd, J = 17.4, 2.8 Hz, 1
H), 2.53 (dd, J = 17.4, 9.2 Hz, 1 H), 2.20 (s, 3 H), 1.68 (m, 1 H), 0.94
(d, J = 6.8 Hz, 3 H), 0.91 (d, J = 6.8 Hz, 3 H); 13C NMR (100 MHz,
CDCl3) δ 208.3, 159.1, 130.8, 129.3, 113.7, 80.0, 71.8, 55.2, 45.0, 31.2,
31.0, 18.3, 17.5; [α]26 = −32.5 (c 1.10, CHCl3); IR (neat) 3438,
D
2962, 2877, 1713, 1362, 1065 cm−1; HRFABMS calcd for C15H23O3
[M + H]+ 251.1642, found 251.1605.
(E)-(S)-5-(4-Methoxybenzyloxy)-3,6-dimethylhept-2-en-1-ol
((E)-14) and (Z)-(S)-5-(4-Methoxybenzyloxy)-3,6-dimethylhept-
2-en-1-ol ((Z)-14). To a solution of 15 (3.60 g, 14.4 mmol) in THF
(40 mL) was added vinylmagnesium bromide (29 mL, 2.0 M in THF,
28.8 mmol) at 0 °C under argon. After being stirred at the same
temperature for 20 min, the mixture was quenched with saturated
aqueous NH4Cl, and the aqueous layer was extracted with ethyl
F
dx.doi.org/10.1021/jo501130b | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
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4.8, 1.6 Hz, 1 H), 2.23−1.97 (m, 2 H), 1.47 (ddd, J = 14.0, 8.4, 5.6 Hz,
1 H), 1.32 (ddd, J = 14.0, 8.4, 4.0 Hz, 1 H), 0.97 (d, J = 6.8 Hz, 3 H),
0.91 (d, J = 6.8 Hz, 3 H), 0.89 (d, J = 6.8 Hz, 3 H); 13C NMR (100
MHz, CDCl3) δ 202.9, 159.1, 131.0, 129.5, 113.8, 80.6, 70.1, 55.3,
50.3, 36.8, 29.8, 25.0, 21.1, 18.2, 17.1; [α]24D = −30.2 (c 1.05, CHCl3);
IR (neat) 2872, 1725, 1612, 1514, 1248, 1067, 1036, 821 cm−1;
HRFABMS calcd for C17H27O3 [M + H]+ 279.1960, found 279.1920.
(3R,4S,6S,8S)-8-(4-Methoxy-benzyloxy)-3,6,9-trimethyldec-
1-en-4-ol (11). To a suspension of aldehyde 13 (691 mg, 2.4 mmol)
and molecular sieves 4 Å (691 mg) in toluene (4.8 mL) was added a
solution of crotylborane (E)-12 (12.7 mL, ca. 0.39 M solution in
toluene, ca. 4.9 mmol) at −78 °C dropwise over 50 min. After being
stirred at the same temperature for 4 h, the reaction mixture was
quenched with aqueous NaOH (7.0 mL, 2 M) and stirred at 0 °C for
50 min. The aqueous layer was extracted with ethyl acetate. The
combined organic layers were dried over MgSO4 and concentrated in
vacuo. The residue was purified by silica gel column chromatography
(5% ethyl acetate/hexane) to give 11 and its diastereomer at a ratio of
9:1 (combined yield 779 mg, 97%) as colorless oil. The mixture of the
diastereomers was partially purified by preparative RP-HPLC (column,
YMC-Pack R&D ODS-A 20 mm × 150 mm; flow rate, 10.0 mL/min;
elution method, H2O/MeOH = 30:70−5:95 linear gradient (0.0−10.0
min), H2O/MeOH = 5:95 isocratic (10.0−15.0 min); retention time,
13.1 min) to afford pure 11, whose structure was determined by
instrumental analyses: 1H NMR (600 MHz, CDCl3) δ 7.28 (d, J = 8.9
Hz, 2 H), 6.86 (d, J = 8.2 Hz, 2 H) 5.73 (ddd, J = 17.1, 10.9, 8.2 Hz, 1
H), 5.13−5.06 (m, 2 H), 4.48 (d, J = 10.9 Hz, 1 H), 4.42 (d, J = 10.9
Hz, 1 H), 3.80 (s, 3 H), 3.47 (br. s., 1 H), 3.29 (dt, J = 8.7, 4.2 Hz, 1
H), 2.19−2.07 (m, 1 H), 2.01−1.82 (m, 2 H), 1.53−1.40 (m, 2 H),
1.35−1.28 (m, 1 H), 1.14 (ddd, J = 14.0, 9.9 2.1 Hz, 1 H), 1.01 (d, J =
6.8 Hz, 3 H), 0.95−0.86 (m, 9 H); 13C NMR (150 MHz, CDCl3) δ
159.0, 140.6, 131.2, 129.5, 116.0, 113.7, 81.1, 72.5, 71.0, 55.3, 45.0,
41.1, 38.1, 30.2, 26.5, 20.3, 18.1, 17.5, 16.1; IR (CHCl3) 2958, 2932,
2871, 2362, 1612, 1514, 1464, 1375, 1302, 1248, 1173, 1066, 1051,
1037, 911, 821, 757 cm−1; [α]25D −31.8 (c 0.055, CHCl3); HRESIMS
calcd for C21H34O3Na [M + Na]+ 357.2400, found 357.2391.
trichlorobenzoyl chloride (1.06 mL, 6.78 mmol) at 0 °C under argon.
The solution was stirred at the same temperature for 10 min. To the
resultant mixture were added a solution of the crude alcohol in toluene
(23 mL) and 4-(dimethylamino)pyridine (0.96 g, 7.91 mmol) at 0 °C
under argon. After being stirred at room temperature for 30 min, the
reaction mixture was quenched with saturated aqueous NaHCO3, and
the aqueous layer was extracted with ethyl acetate. The combined
organic layers were dried over MgSO4 and concentrated in vacuo. The
residue was purified by silica gel column chromatography (10% to 15%
ethyl acetate/hexane) to give 20 (1.47 g, 2.06 mmol, 91% in 2 steps)
as a colorless oil: 1H NMR (400 MHz, CDCl3, mixture of rotamers) δ
7.84−7.69 (m, 2 H), 7.66−7.51 (m, 2 H), 7.43−7.35 (m, 2 H), 7.34−
7.27 (m, 2 H), 5.80−5.58 (m, 1 H), 5.13−4.95 (m, 2 H), 4.93−4.79
(m, 2 H), 4.79−4.67 (m, 2 H), 4.50−4.14 (m, 4 H), 3.72−3.45 (m, 2
H), 2.52−2.16 (m, 2 H), 2.16−1.87 (m, 3 H), 1.86−1.07 (m, 6 H),
1.06−0.90 (m, 4.5 H), 0.90−0.79 (m, 7.5 H) ; 13C NMR (100 MHz,
CDCl3) δ 172.5, 172.3, 154.7, 154.4, 154.17, 154.15, 144.25, 144.23,
144.0, 143.8, 141.35, 141.31, 141.26, 138.8, 138.7, 127.71, 127.69,
127.67, 127.13, 127.08, 127.05, 127.02, 125.4, 125.23, 125.16, 119.96,
119.95, 116.3, 94.8, 80.5, 80.3, 77.09, 77.06, 76.58, 76.56, 67.8, 67.3,
59.6, 59.4, 47.2, 47.0, 46.3, 42.35, 42.33, 38.54, 38.43, 37.98, 37.80,
31.2, 30.9, 29.9, 26.2, 26.1, 24.3, 23.3, 19.3, 19.2, 18.5, 18.4, 16.63,
16.57, 15.5, 15.4; IR (CHCl3) 2963, 2362, 2357, 2342, 1754, 1707,
1451, 1417, 1379, 1348, 1249, 197, 1120, 1087, 989, 944, 923, 821,
758, 740 cm−1; [α]25 −45.6 (c 1.86, CHCl3); HRESIMS calcd for
D
C36H44NO7Cl3Na [M + Na]+ 730.2076, found 730.2056.
Pro-Dtrina 9. To a solution of 20 (111 mg, 0.157 mmol) in DMF
(1.5 mL) were added OsO4 (0.031 mL, 0.05 M solution in THF, 1.57
μmol), oxone (0.386 g, 0.628 mmol), and NaHCO3 (52.7 mg, 0.628
mmol) at room temperature. After being stirred at the same
temperature for 23 h, the reaction mixture was diluted with H2O
(3.0 mL) and 2-methyl-2-propanol (1.57 mL). To the mixture was
added NaIO4 (0.067 mg, 0.314 mmol) at room temperature. The
resulting mixture was stirred at the same temperature for 6 h and
poured into aqueous HCl (1 M) and CH2Cl2. The aqueous layer was
extracted with CH2Cl2. The combined organic layers were washed
with an aqueous solution of Na2S2O3 (10 wt %) and brine, dried over
MgSO4, and concentrated in vacuo. The residue was purified by silica
gel column chromatography (40% ethyl acetate/hexane) to give 9
(3R,4S,6S,8S)-5-(4-Methoxybenzyloxy)-3,6,9-trimethyl-1-en-
4-yl 2,2,2-trichloroethyl Carbonate (19). To a solution of 11
(0.758 g, 2.26 mmol) and pyridine (0.54 mL, 6.78 mmol) in CH2Cl2
(11 mL) were added 2,2,2-trichloroethoxycarbonyl chloride (0.37 mL,
2.71 mmol) and 4-dimethylaminopyridine (13 mg, 0.11 mmol) at 0
°C. After being stirred at the same temperature for 2 h, the reaction
mixture was quenched with saturated aqueous NH4Cl, and the
aqueous layer was extracted with ethyl acetate. The combined organic
layers were dried over MgSO4, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (5% ethyl acetate/
hexane) to give carbonate 19 and its diastereomer (1.15 g, 2.26 mmol,
1
(87.9 mg, 0.121 mmol, 77%) as a white amorphous solid: H NMR
(400 MHz, CDCl3, mixture of rotamers) δ 7.80−7.72 (m, 2 H), 7.66−
7.53 (m, 2 H), 7.44−7.35 (m, 2 H), 7.35−7.28 (m, 2 H), 5.23−5.04
(m, 1 H), 4.95−4.84 (m, 1 H), 4.83−4.69 (m, 2 H), 4.56−4.13 (m, 4
H), 3.70−3.44 (m, 2 H), 2.99−2.77 (m, 1 H), 2.39−2.16 (m, 1 H),
2.15−1.64 (m, 6 H), 1.63−1.31 (m, 3 H), 1.29−1.09 (m, 3 H) 1.04−
0.93 (m, 1.5 H), 0.86 (d, J = 5.4 Hz, 7.5 H) ; 13C NMR (100 MHz,
CDCl3) δ 172.6, 172.1, 155.1, 154.5, 153.78, 153.74, 144.20, 144.11,
143.89, 143.82, 141.4, 127.7, 127.1, 125.40, 125.26, 125.21, 125.15,
120.0, 94.65, 94.62, 77.7, 77.2, 76.9, 67.8, 67.6, 59.5, 59.4, 47.18, 47.12,
47.04, 47.0, 46.4, 43.2, 43.0, 38.7, 37.8, 37.7, 37.3, 37.0, 31.3, 31.2,
31.0, 30.0, 25.9, 25.7, 24.2, 23.8, 23.3, 20.2, 19.3, 19.1, 18.4, 18.2, 17.1,
16.7, 11.9, 11.7, 11.5; IR (CHCl3) 2962, 1757, 1741, 1708, 1452, 1420,
1
quant) as a colorless oil: H NMR (400 MHz, CDCl3) δ 7.27 (d, J =
8.3 Hz, 2 H), 6.87 (d, J = 8.3 Hz, 2 H), 5.79−5.66 (m, 1 H), 5.12−
5.01 (m, 2 H), 4.89−4.82 (m, 1 H), 4.77 (d, J = 12.7 Hz, 1 H), 4.64
(d, J = 13.2 Hz, 1 H), 4.47 (d, J = 11.2 Hz, 1 H), 4.42 (d, J = 11.7 Hz,
1.0 H), 3.79 (s, 3 H), 3.23 (dt, J = 8.1, 3.8 Hz, 1 H), 2.49−2.37 (m, 1
H), 1.98−1.87 (m, 1 H), 1.80−1.68 (m, 2 H), 1.44 (ddd, J = 13.9, 8.3,
5.6 Hz, 1 H), 1.32−1.22 (m, 1 H), 1.10−1.20 (m, 1 H), 1.04 (d, J =
6.8 Hz, 3 H), 0.95 (d, J = 6.3 Hz, 3 H), 0.91−0.85 (m, 6 H); 13C NMR
(100 MHz, CDCl3) δ 159.1, 154.3, 139.0, 131.3, 129.3, 116.2, 113.8,
94.8, 80.9, 80.7, 76.6, 71.1, 55.2, 42.5, 38.3, 37.9, 30.2, 25.9, 20.2, 18.0,
17.4, 15.6; IR (CHCl3) 2959, 2363, 2359, 1756, 1613, 1513, 1466,
1380, 1248, 1065, 1038, 923, 820, 777, 733 cm−1; HRESIMS calcd for
C24H35O5Cl3Na [M + Na]+ 531.1442, found 531.1433.
Prolyl Ester 20. To a solution of 19 (1.53 g, 2.26 mmol) in
CH2Cl2 (20 mL) and H2O (2.0 mL) was added 2,3-dichloro-5,6-
dicyanobenzoquinone (1.03 g, 4.52 mmol) at 0 °C. After being stirred
at the same temperature for 1 h, the reaction mixture was quenched
with saturated aqueous NaHCO3, and the aqueous layer was extracted
with CHCl3. The combined organic layers were dried over MgSO4 and
concentrated in vacuo. The residue was used for the next reaction
without further purification.
1378, 1352, 1248, 1199, 1121, 1090, 942, 819, 758, 740 cm−1; [α]26
D
−35.7 (c 1.49, CHCl3); HRESIMS calcd for C35H42NO9Cl3Na [M +
Na]+ 748.1817, found 748.1796.
Amide 21. To a solution of 1012,13 (119 mg, 0.219 mmol) in
CH2Cl2 (0.8 mL) were added a solution of 9 (123 mg, 0.169 mmol) in
CH2Cl2 (0.8 mL), DIEA (0.117 mL, 0.67 mmol), HOAt (27.0 mg,
0.202 mmol), and EDCI·HCl (38.7 mg, 0.202 mmol) at 0 °C under
argon. After being stirred at the same temperature for 12 h, the
reaction mixture was diluted with ethyl acetate and poured into
aqueous HCl (1 M) at 0 °C. The organic layer was separated, and the
aqueous layer was extracted with ethyl acetate. The combined organic
layers were washed with brine, dried over MgSO4, and concentrated in
vacuo. The residue was purified by silica gel column chromatography
(15% to 30% ethyl acetate/hexane) to give 21 (106 mg, 0.092 mmol,
55%) as white amorphous solid: 1H NMR (600 MHz, CDCl3, mixture
of rotamers) δ 7.77−7.75 (m, 2 H), 7.70−7.68 (m, 0.4H), 7.58 (m, 1.6
H), 7.41−7.19 (m, 19 H), 6.39 (dd, J = 9.0, 1.2 Hz, 0.6 H), 6.32 (dd, J
= 9.0, 1.8 Hz, 0.4 H), 6.25 (brd, J = 4.2 Hz, 0.6 H), 5.98−5.83 (m, 1
To a solution of N-Fmoc-L-proline (1.65 g, 6.78 mmol) in toluene
(23 mL) was added DIEA (1.18 mL, 6.78 mmol) and 2,4,6-
G
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H), 5.35−5.17 (m, 2.4 H), 5.01−4.95 (m, 1 H), 4.90−4.83 (m, 1 H),
4.82 (d, J = 12.0 Hz, 0.6 H), 4.73 (d, J = 12.0 Hz, 0.4 H), 4.63−4.61
(m, 2 H), 4.54 (d, J = 5.4 Hz, 1 H), 4.48−4.23 (m, 5 H), 3.65−3.50
(m, 2 H), 2.51−2.29 (m, 2.7 H), 2.20−1.93 (m, 4.3 H), 1.77−1.70 (m,
2 H), 1.73 (s, 1.8 H), 1.69 (s, 1.2 H), 1.68−1.63 (m, 0.6 H), 1.52−
1.44 (m, 2.4 H), 1.15- 1.10 (m, 1 H), 1.09 (d, J = 7.0 Hz, 1.8 H), 0.98
(d, J = 7.0 Hz, 1.2 H), 0.92 (d, J = 7.0 Hz, 1.8 H), 0.87−0.82 (m, 6 H),
0.81 (d, J = 7.0 Hz, 1.2 H); 13C NMR (150 MHz, CDCl3) δ 172.4,
172.2, 171.7, 167.2, 154.8, 154.6, 153.8, 153.7, 144.5, 144.4, 144.1,
143.9, 143.8, 139.4, 139.2, 132.2, 130.4, 130.2, 129.6, 128.05, 128.04,
127.7, 127.2, 126.9, 126.8, 125.7, 125.4, 125.14, 125.09, 119.97, 118.2,
94.8, 79.0, 78.4, 76.5, 76.4, 67.9, 67.5, 67.2, 67.0, 65.5, 65.4, 59.6, 59.3,
47.2, 47.0, 46.4, 45.1, 44.8, 38.5, 38.3, 37.5, 37.0, 36.0, 35.7, 31.73,
31.67, 31.2, 31.1, 30.0, 25.9, 25.5, 24.3, 23.4, 19.6, 19.3, 18.4, 18.3,
17.0, 13.4, 13.0, 12.9; IR (CHCl3) 2964, 2960, 2362, 2359, 2342, 2332,
1758, 1710, 1679, 1451, 1447, 1419, 1248, 1198, 1182, 1121, 1087,
757, 742, 701, 668 cm−1; [α]26D −24 (c 1.2, CHCl3); HRFABMS calcd
After being stirred at 0 °C to room temperature for 80 min, the
reaction mixture was concentrated in vacuo. The residue was purified
by silica gel column chromatography (10% to 30% acetone/hexane) to
1
afford 23 (45 mg, 41 μmol, 95%) as a white amorphous solid. H
NMR (600 MHz, CD2Cl2, mixture of rotamers) δ 7.78 (m, 2 H),
7.69−7.56 (m, 2 H), 7.40 (m, 2 H), 7.32 (m, 2 H), 7.15−7.04 (m, 2
H), 6.82−6.73 (m, 2 H), 6.63−6.43 (m, 1 H), 6.32−6.21 (m, 1 H),
5.98−5.85 (m, 1 H), 5.50−5.00 (m, 5 H), 4.96−4.85 (m, 2 H), 4.62−
4.54 (m, 2 H), 4.49−4.19 (m, 3 H), 3.80−3.69 (m, 4 H), 3.67−3.30
(m, 4 H), 3.08−2.76 (m, 6 H), 2.75−2.68 (m, 3 H), 2.34−2.20 (m, 1
H), 2.14−1.44 (m, 15 H), 1.43−1.14 (m, 10 H), 1.12−0.73 (m, 12
H); 13C NMR (150 MHz, CD2Cl2, mixture of rotamers) δ 173.0,
172.2, 171.7, 171.0, 168.3, 159.2, 155.4, 154.7, 145.1, 144.9, 144.7,
144.5, 141.8, 132.6, 131.1, 130.1, 128.9, 128.2, 127.6, 126.0, 125.8,
125.8, 120.5, 118.8, 114.3, 114.2, 77.5, 76.7, 72.0, 68.2, 68.1, 65.8, 61.1,
60.3, 60.0, 55.7, 51.2, 50.3, 47.8, 47.8, 47.2, 46.2, 41.6, 40.3, 39.9, 38.2,
33.7, 32.8, 32.1, 31.7, 31.4, 31.4, 31.0, 30.9, 30.5, 26.2, 25.7, 25.5, 25.1,
23.9, 20.2, 20.1, 18.9, 17.8, 17.4, 16.9, 16.1, 14.6, 13.8, 10.8; IR
(CH2Cl2) 2963, 2932, 2877, 1739, 1706, 1652, 1646, 1634, 1513,
+
for C63H70Cl3N2O10S [M + H] 1151.3817, found 1151.3807.
Thiazoline 22. To a solution of Ph3PO (425 mg, 1.56 mmol) in
CH2Cl2 (3 mL) was added Tf2O (131 μL, 0.78 mmol) at 0 °C under
argon, and the mixture was stirred at the same temperature for 30 min.
The resulting mixture (600 μL) was added slowly to a solution of 21
(60 mg, 52.1 μmol) in CH2Cl2 (1.5 mL) at 0 °C under argon. After
the mixture was stirred at the same temperature for 15 min, the
reaction mixture was quenched with saturated aqueous NaHCO3 at 0
°C, and the aqueous layer was extracted with ethyl acetate. The
combined organic layers were washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was used for the next reaction
without further purification.
1478, 1464, 1452, 1417, 1249, 1180, 1122, 990, 759, 740 cm−1; [α]25
D
−1.2 × 102 (c 0.37, CH2Cl2); HRESIMS calcd for C62H83N5O11SNa
[M + Na]+ 1128.5702, found 1128.5670.
Apratoxin C (3). To a solution of allyl ester 23 (55 mg, 0.049
mmol) and N-methylaniline (0.013 mL, 0.122 mmol) in THF (5 mL)
was added a catalytic amount of Pd(PPh3)4 (5.6 mg, 0.0049 mmol) at
0 °C under argon. After being stirred at 0 °C to room temperature for
2 h, the reaction mixture was concentrated in vacuo. The residue was
passed through a pad of silica gel (20% to 50% ethyl acetate/hexane,
5% MeOH/CH2Cl2) to afford the crude carboxylic acid, and the
resultant acid was used for next reaction without further purification.
To a solution of the carboxylic acid in MeCN (2.0 mL) was added
diethylamine (1.0 mL) at 0 °C under argon. After being stirred at 0 °C
to room temperature for 1 h, the reaction mixture was concentrated in
vacuo. The residue was azeotroped with CH2Cl2 twice and then
dissolved in CH2Cl2 (49 mL). To this solution were added DIEA
(0.076 mL, 0.44 mmol) and HATU (56 mg, 0.15 mmol) at 0 °C under
argon. After being stirred at 0 °C to room temperature for 18 h, the
reaction mixture was concentrated in vacuo. The residue was purified
by silica gel column chromatography (50% to 100% hexane/ethyl
acetate) and subsequent reversed-phase HPLC (column, YMC-Pack
R&D ODS-A 20 mm × 150 mm; flow rate, 10.0 mL/min; elution
method, H2O/MeOH = 20:80−0:100 linear gradient (0.0−10.0 min),
H2O/MeOH = 0:100 isocratic (10.0−15.0 min); retention time, 10.5
min) to give apratoxin C (3) (8.5 mg, 0.010 mmol, 21%) as a white
amorphous solid: 1H NMR (600 MHz, CDCl3) δ 7.15 (d, J = 8.6 Hz,
2 H), 6.80 (d, J = 8.6 Hz, 2 H), 6.35 (d, J = 9.6 Hz, 1 H), 6.06 (d, J =
9.6 Hz), 5.24 (ddd, J = 9.0, 8.6, 4.2 Hz, 1 H), 5.19 (d, J = 11.4 Hz, 1
H), 5.05 (ddd, J = 10.8, 8.4, 4.8 Hz, 1 H), 4.99 (ddd, J = 12.0, 6.6, 2.4
Hz, 1 H), 4.66 (d, J = 10.2 Hz, 1 H), 4.22 (m, 1 H), 4.18 (t, J = 7.8 Hz,
1 H), 3.78 (s, 3 H), 3.67 (m, 1 H), 3.55 (dddd, J = 10.2, 10.2, 10.2, 1.8
Hz, 1 H), 3.46 (dd, J = 10.2, 9.8 Hz, 1 H), 3.30 (brq, J = 7.2 Hz, 1 H),
3.13 (dd, J = 10.2, 4.2 Hz, 1 H), 3.11 (brt, J = 12.0 Hz, 1 H), 2.86 (dd,
J = 12.0, 6.0 Hz, 1 H), 2.82 (s, 3 H), 2.71 (s, 3 H), 2.64 (m, 1 H), 2.22
(m, 3 H), 2.06 (m, 1 H), 1.96 (brs, 3 H), 1.89 (m, 2 H), 1.77 (ddd, J =
14.3, 12.0, 3.0 Hz 1 H), 1.71 (m, 1 H), 1.54 (ddd, J = 10.8, 10.8, 4.2
Hz, 1 H), 1.29 (m, 2 H), 1.22 (d, J = 7.2 Hz, 3 H), 1.12 (ddd, J = 13.8,
11.4, 2.4 Hz, 1 H), 1.07 (d, J = 7.2 Hz, 3 H), 0.98 (d, J = 6.6 Hz, 3 H),
0.95 (m, 1 H), 0.92 (m, 6 H), 0.89 (d, J = 7.2 Hz, 3 H), 0.86 (d, J = 7.2
Hz, 3 H); 13C NMR (150 MHz, CDCl3) δ 177.4, 172.8, 170.53,
170.47, 170.0, 169.6, 158.6, 136.3, 130.6, 130.5, 128.2, 113.9, 75.2,
72.5, 71.6, 60.6, 59.7, 56.8, 55.3, 50.4, 49.1, 47.7, 40.4, 38.2, 37.6, 37.1,
36.7, 33.2, 31.7, 30.4, 29.2, 25.6, 24.6, 24.2, 19.7, 18.8, 18.0, 16.6, 14.0,
13.9, 13.3, 9.1; IR (solid) 3418, 2961, 2931, 2873, 1734, 1623, 1507,
To a solution of the crude thiazoline in THF (1.6 mL) and aqueous
1 M NH4Cl (800 μL) was added Zn dust (68.1 mg, 1.04 mmol) at
room temperature. After being stirred for 1.5 h at the same
temperature, the mixture was partitioned between ethyl acetate and
brine. The aqueous layer was extracted with ethyl acetate, and the
combined organic layers were dried over Na2SO4 and concentrated in
vacuo. The residue was purified by silica gel column chromatography
(30% ethyl acetate/hexane) to afford 22 (33.6 mg, 46.9 μmol, 90%) as
1
a white amorphous solid: H NMR (600 MHz, CD2Cl2, mixture of
rotamers) δ 7.78 (d, J = 7.6 Hz, 2 H), 7.69−7.56 (m, 2 H), 7.43−7.37
(m, 2 H), 7.35−7.27 (m, 2 H), 6.80−6.71 (m, 1 H), 6.01−5.87 (m, 1
H), 5.31−5.15 (m, 3 H), 4.96−4.88 (m, 1 H), 4.66−4.55 (m, 2 H),
4.48−4.19 (m, 4 H), 3.76−3.33 (m, 4 H), 3.00−2.90 (m, 1 H), 2.71−
2.50 (m, 1 H), 2.34−2.20 (m, 1 H), 2.15−1.82 (m, 7 H), 1.82−1.47
(m, 4 H), 1.22−1.17 (m, 3 H), 1.08−1.00 (m, 1 H), 0.96−0.77 (m, 9
H); 13C NMR (150 MHz, CD2Cl2) δ 173.3, 173.0, 167.6, 155.4, 154.7,
145.1, 144.9, 144.7, 144.5, 141.9, 141.8, 141.7, 133.1, 133.0, 128.2,
128.2, 127.6, 127.6, 126.0, 125.9, 125.8, 125.6, 120.5, 120.4, 118.2,
118.2, 77.6, 76.6, 72.0, 71.9, 68.2, 68.1, 66.0, 65.9, 65.9, 60.2, 60.1,
47.8, 47.7, 47.5, 47.1, 46.3, 45.6, 41.6, 40.3, 39.9, 39.7, 38.1, 32.8, 32.3,
32.1, 31.7, 30.6, 30.5, 30.3, 30.2, 26.3, 25.7, 25.1, 23.9, 20.7, 20.3, 20.1,
19.0, 18.9, 17.8, 17.4, 17.4, 16.8, 16.1, 13.5, 13.5; IR (CH2Cl2) 2964,
2359, 2342, 2328, 1739, 1734, 1714, 1684, 1457, 1451, 1437, 1423,
1419, 1245, 1199, 1185, 1120, 740, 669 cm−1; [α]22 −64 (c 0.32,
D
CH2Cl2); HRFABMS calcd for C41H53N2O7S [M + H]+ 717.3573,
found 717.3563.
Cyclization Precursor 23. To a solution of allyl ester 22 (31 mg,
43 μmol) and N-methylaniline (11.6 μL, 107 μmol) in THF (2.0 mL)
was added a catalytic amount of Pd(PPh3)4 (4.9 mg, 4.3 μmol) at 0 °C
under argon. After being stirred at 0 °C to room temperature for 30
min, the reaction mixture was concentrated in vacuo. The residue was
passed through a pad of silica gel (20% to 100% ethyl acetate/hexane)
to afford the crude carboxylic acid, and the resultant acid was used for
the next reaction without further purification.
To a solution of tripeptide 812,13 (57 mg, 86 μmol) in MeCN (3.4
mL) was added diethylamine (1.7 mL) at room temperature. After
being stirred at the same temperature for 20 min, the reaction mixture
was concentrated in vacuo. The residue was azeotroped with CH2Cl2
twice, and then dissolved in CH2Cl2 (1 mL). This solution was added
to the solution of the carboxylic acid, DIEA (15 μL, 86 μmol) and
HATU (16 mg, 43 μmol) in CH2Cl2 (1 mL) at 0 °C under argon.
1457, 1248, 1181 cm−1; [α]28 −1.8 × 102 (c 0.43, MeOH) [lit.5
D
[α]25 = −171 (c 0.22, MeOH)]; HRFABMS calcd for C44H68N5O8S
D
[M + H]+ 826.4789, found 826.4810.
1H NMR (600 MHz, CD3CN) δ 7.17 (d, J = 8.3 Hz, 2 H), 6.84 (d, J
= 8.3 Hz, 2 H), 6.60 (brs, 1 H), 6.14 (m, 1 H), 5.26, (ddd, J = 10.3,
8.7, 2.8 Hz, 1 H), 5.09 (d, J = 11.7 Hz, 1 H), 4.95 (ddd, J = 12.3, 6.1,
2.5 Hz, 1 H), 4.90 (m, 1 H), 4.45 (d, J = 11.2 Hz), 4.09 (t, J = 7.8 Hz,
H
dx.doi.org/10.1021/jo501130b | J. Org. Chem. XXXX, XXX, XXX−XXX

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Article
1 H), 4.05 (m, 1 H), 3.75 (s, 3 H), 3.59 (m, 1 H), 3.44 (dddd, J = 11.5,
11.2, 10.0, 3.4 Hz, 1 H), 3.41, (dd, J = 11.1, 8.7 Hz, 1 H), 3.40 (brs, 1
H), 3.10 (m, 1 H), 3.03 (ddd, J = 11.1, 2.8, 1.1 Hz, 1 H), 2.89 (m, 1
H), 2.80 (s, 3 H), 2.57 (m, 3 H), 2.55 (dq, J = 10.0, 7.0 Hz, 1 H), 2.25
(m, 1 H), 2.13 (m, 1 H), 2.11 (ddqdd, J = 11.8, 11.7, 6.7, 4.0, 3.2 Hz, 1
H), 2.03 (m, 1 H), 1.92 (s, 3 H), 1.84 (m, 1 H), 1.78 (m, 1 H), 1.74
(ddd, J = 14.3, 12.3, 3.2 Hz, 1 H), 1.67 (m, 1 H), 1.47 (ddd, J = 13.3,
11.5, 4.0 Hz, 1 H), 1.32 (ddd, J = 14.3, 11,8, 2.5 Hz, 1 H), 1.21 (m, 1
H), 1.17 (ddd, J = 13.3, 11.7, 3.4 Hz, 1 H), 1.06 (d, J = 6.8 Hz, 3 H),
1.02 (d, J = 6.9 Hz, 3 H), 0.93 (d, J = 6.7 Hz, 3 H), 0.88 (d, J = 6.8 Hz,
3 H), 0.84 (d, J = 6.8 Hz, 3 H), 0.83 (m, 1 H), 0.83 (m, 3 H), 0.82 (m,
3 H); 13C NMR (150 MHz, CD3CN) δ 176.8, 173.5, 171.4, 170.7,
170.0, 159.5, 136.7, 131.4, 130.3, 129.9, 114.6, 75.7, 73.0, 72.2, 60.6,
57.4, 55.8, 50.0, 48.4, 40.8, 39.1, 38.3, 37.0, 33.9, 32.3, 30.4, 30.0, 26.1,
25.2, 25.0, 19.7, 19.0, 18.2, 17.0, 14.5, 14.3, 13.3, 9.2. (1H and 13C
NMR chemical shift assignments of apratoxin C (3) in CD3CN are
summarized in Table S4 in Supporting Information).
Cytotoxicity Assay. Human colon adenocarcinoma HCT116 cells
were kindly provided by Prof. Yoshiteru Ohshima at the Graduate
School of Pharmaceutical Sciences in Tohoku University. They were
cultured in an RPMI 1640 medium (Gibco, Thermo Fisher Scientific,
Life Technologies) supplemented with 10% fetal bovine serum
(Equitech-Bio, Inc.) at 37 °C under 5% CO2. For the cytotoxicity
assay, near-confluent cultures of the cells were plated at 5 × 103 cells/
100 μL/well in fresh culture medium in a 96-well clear bottom plate
and incubated at 37 °C under 5% CO2 for 24 h before the
experiments.
Apratoxin A (1) or C (3) was dissolved in DMSO at concentrations
ranging from 0.01 to 10 μM. One microliter of the resultant solution
was added to the above-mentioned 100 μL cell culture, resulting in
various concentrations of the compound (0.1−100 nM) or solvent
control (DMSO 1%). After a 48-h incubation at 37 °C under 5% CO2,
10 μL of WST-8 reagent solution (Cell Count Reagent SF, Nacalai
Tesque, Inc.)33,34 was added to the cell culture. The cell culture was
then incubated at 37 °C under 5% CO2 for 2 h. Colorimetric
determination of WST-8 was conducted at 595 nm using a microplate
reader. The absorbance obtained upon the addition of the vehicle was
considered as 100%.
dihedral angle constraints (1H−C−C−1H angle, 180 30°) at 10,000
iterations with 500 times of energy minimization. Then, energy
minimization was conducted on each found structure without
constraints.
H/D exchange experiments were performed on 3.2 mg of apratoxin
C (3) in 0.5 mL of CD3CN with the addition of 10 μL of D2O.
ASSOCIATED CONTENT
■
S
* Supporting Information
Supporting figures and tables, copies of H and 13C NMR
1
spectra for synthetic compounds, and 2D NMR spectra of
apratoxin C (3) and apratoxin A (1). This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: doi_taka@mail.pharm.tohoku.ac.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by Grant-in-Aid for Scientific
Research (B) (nos. 23310145 and 26282208 for T.D.) from
MEXT. The authors are grateful to Prof. Yoichi Nakao at
School of Advanced Science and Engineering at Waseda
1
University for kindly providing the H NMR spectrum of
natural apratoxin C in CD3CN. The authors are grateful to
Prof. Yoshiteru Ohshima and Dr. Teigo Asai at the Graduate
School of Pharmaceutical Sciences in Tohoku University for
allowing us to use their facility for the cell-based assay.
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