Total synthesis of spiruchostatin B aided by an automated synthesizer

Article abstract of DOI:10.1039/c0ob01169j

The total synthesis of a natural product HDAC inhibitor, spiruchostatin B, was successfully achieved. A 5-step synthesis that included an asymmetric aldol reaction was carried out in an automated synthesizer to provide an (E)-(S)-3-hydroxy-7-thio-4-heptenoic acid segment that is the crucial structure of cysteine-containing, depsipeptidic natural products such as spiruchostatins, FK228, FR901375, and largazole for their inhibitory activity against HDACs.

Full text of DOI:10.1039/c0ob01169j

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PAPER
Total synthesis of spiruchostatin B aided by an automated synthesizer†
Shinichiro Fuse,a Kumiko Okada,a Yusuke Iijima,a Asami Munakata,b Kazuhiro Machida,c
Takashi Takahashi,*a Motoki Takagi,b Kazuo Shin-yad and Takayuki Doi*e
Received 13th December 2010, Accepted 28th February 2011
DOI: 10.1039/c0ob01169j
The total synthesis of a natural product HDAC inhibitor, spiruchostatin B, was successfully achieved.
A 5-step synthesis that included an asymmetric aldol reaction was carried out in an automated
synthesizer to provide an (E)-(S)-3-hydroxy-7-thio-4-heptenoic acid segment that is the crucial
structure of cysteine-containing, depsipeptidic natural products such as spiruchostatins, FK228,
FR901375, and largazole for their inhibitory activity against HDACs.
Introduction
HDAC (histone deacetylase) inhibitors make up a very important
class of compounds because they show promise as prospective
targets for cancer chemotherapy.1 There are 18 HDACs divided
into four main sub-types: classes I, II, IV, and the structurally
distinct class III.2 HDAC inhibitor design has concentrated on the
class I and class II zinc-dependent enzymes.3 Although the precise
targets of class I and class II HDACs remain uncertain, class I
selectivity is now being considered useful for anticancer agents,4
whereas the inhibition of specific class II HDACs is considered
to promote undesired cardiac hypertrophy.5 Synthetic HDAC
inhibitors, such as hydroxamates and short-chain fatty acids, suffer
from relatively weak HDAC inhibition (micromolar IC50) and
lack of class I selectivity.3 On the other hand, cysteine-containing,
depsipeptidic natural products including spiruchostatin A (1), B
(2),6 FK228,7 FR901375,8 and largazole9 have a high potency
and a class I selectivity. All these compounds retain (E)-(S)-3-
hydroxy-7-thio-4-heptenoic acid (emphasized in Scheme 1) that is
a crucial structure for inhibitory activity.10 The development of an
efficient synthetic method for the discovery of more potent and
class I selective analogues of HDAC inhibitors is very important.
Recently, the successful total synthesis of the natural product
HDAC inhibitors and their analogues and biological evaluation
aDepartment of Applied Chemistry, Tokyo Institute of Technology, 2-
12-1, Ookayama, Meguro-ku, Tokyo, 152-8552, Japan. E-mail: ttak@
apc.titech.ac.jp; Fax: +81-3-5734-2884; Tel: +81-3-5734-2120
bJapan Biological Informatics Consortium (JBIC), 2-4-7, Aomi, Koto-ku,
Tokyo, 135-0064, Japan
cDevelopment Department, ChemGenesis Incorporated; 4-10-1, Nihonbashi-
honcho, Chuo-ku, Tokyo, 103-0023, Japan
dNational Institute of Advanced Industrial Science and Technology, 2-4-7,
Aomi, Koto-ku, Tokyo, 135-0064, Japan
Scheme
products and the synthetic strategy for spiruchostatin B (2). The
(E)-(S)-3-hydroxy-7-thio-4-heptenoic acid moiety that is crucial
structure for HDAC inhibition is emphasized. Trt = trityl, Boc =
t-butoxycarbonyl, Fmoc = 9-fluorenylmethoxycarbonyl.
1
Structures of cysteine-containing, depsipeptidic natural
eGraduate School of Pharmaceutical Sciences, Tohoku University, 6-3
Aza-Aoba, Aramaki, Aoba, Sendai, 980-8578, Japan. E-mail: doi_taka@
mail.pharm.tohoku.ac.jp; Fax: +81-22-795-6864; Tel: +81-22-795-6865
a
† Electronic supplementary information (ESI) available: Full picture of
ChemKonzert and detailed protocol to use ChemKonzert. Copies of NMR
spectra of compounds 2–4, 7, 10–12, 13a, 15, 16a, 17–20 are available. See
DOI: 10.1039/c0ob01169j
on the basis of spiruchostatin A (1),11–17 B (2),17,18 FK228,17,19–25
FR901375,26 and largazole25,27–39 have been reported.40 An efficient
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supply of a sufficient amount of (E)-(S)-3-hydroxy-7-thio-4-
heptenoic acid Segment 7 is required for the synthesis of various
analogues of HDAC inhibitors.
Automation of synthetic procedures improves both the re-
producibility and reliability of the syntheses because auto-
mated synthesizers minimize the variability of the experimental
manipulations.41 We previously reported a formal total synthesis
of taxol using an automated synthesizer that was developed in our
R 42,43
laboratory. Use of the ChemKonzertꢀ
enabled production of a
key synthetic intermediate in a 36-step, solution-phase synthesis.44
We also reported an efficient synthesis of a cyclic ether key interme-
diate for 9-membered masked enediyne using the ChemKonzert.45
If synthetic chemists run the reactions in automated synthesizers
and digitally store the experimental procedures, anyone could
reproduce the results by using the same apparatus and compounds,
regardless of the time and place. As a result, synthetic chemists
could expend more effort on the advanced and challenging work
of construction of new synthetic routes.
Scheme 2 Synthesis of b-hydroxy acid 7 aided by an automated synthe-
sizer. Reagents and Conditions: a) TrtSH, Et3N, CH2Cl2, 25 ◦C, 1 h; b)
monoethyl malonate, DMAP, DMF, 100 ◦C, 2 h, 2 steps 88%; c) DIBAL,
CH2Cl2, -20 ◦C to 25 ◦C, 2 h, 75%; d) (COCl)2, DMSO, Et3N, CH2Cl2,
-78 to 0 ◦C, 1 h, 56%; e) n-BuLi, Cp2ZrCl2, THF, -78 to 0 ◦C, 1 h,
84% (13a:13b = 90 : 10); f) NaOH, THF–MeOH–H2O, 0 ◦C, 1 h, 92%.
DMAP = 4-(dimethylamino)pyridine, DMF = N,N-dimethylformamide,
DIBAL = diisobutylaluminium hydride, DMSO = dimethyl sulfoxide,
THF = tetrahydrofuran.
Herein, we wish to report the total synthesis of spiruchostatin
B (2). b-Hydroxy acid segment 7, which is a common synthetic in-
termediate for cysteine-containing, depsipeptidic natural products
were efficiently supplied with the aid of an automated synthesizer.
Results and discussion
The synthetic route for spiruchostatin B (2) is illustrated in
Scheme 1. Macrolactonization of seco-acid derivative 3 and
subsequent disulfide formation is the most crucial step in the
construction of the 15-membered bicyclic skeleton of 2. Com-
pound 3 can be prepared by the sequential coupling of D-allo-
isoleucine-derived (3S, 4R)-statine allyl ester 4, N-Fmoc-S-trityl-
D-cysteine (5), N-Fmoc-D-alanine (6), and b-hydroxy acid 7.12,13
For the preparation of b-hydroxy acid 7, the key step is an
asymmetric aldol reaction. Since acetate aldols proceed with
poor diastereoselectivity with the classical Evans’ oxazolidin-2-
one auxiliary, we have investigated the potential of Seebach’s N-
acetyl-oxazolidin-2-one A46–49 (Scheme 2) and observed that the
Zr-enolate50–52 of 13 was effective in this reaction.12 The developed
asymmetric aldol reaction requires careful synthetic manipulation
to obtain a high yield and a high stereoselectivity. Therefore, we
planned to utilize an automated synthesizer42,43 to improve both
efficiency and reproducibility.44,45
b-Hydroxy acid 7 was prepared with the use of the automated
synthesizer ChemKonzert (Scheme 2).42 Preparation of 10 from
acrolein (8) was carried out as follows. The computer controlling
the automated synthesizer was programmed with a specific
procedure. Reagents, substrate and solvent were added to the
reaction vessel, RF1, the reagent reservoirs, RR1 and RR2, and the
solvent bottle, RS1 (Fig. 1). Then, the operation of the automated
synthesizer was started. TrtSH was stirred at 25 ◦C under a
nitrogen atmosphere in the reaction vessel, RF1. CH2Cl2, Et3N
and acrolein (8) in the solvent bottle, RS1, and reagent reservoirs
RR1 and RR2 were added to the reaction vessel, RF1 (Fig. 1).26
After stirring at 25 ◦C for 1 h, the resultant mixture was transferred
to a round flask, CF1. The entire apparatus was washed with water
and acetone from the solvent tanks, WT1 and WT2, then dried
under reduced pressure (Fig. 1). The collected solution in CF1
was manually concentrated in vacuo. The residue was used for the
next reaction without further purification. Reagents, substrate,
Fig. 1 Schematic diagram of the automated synthesizer, ChemKonzert.
solvent and wash solutions were added to the reaction vessel,
RF1, reagent reservoirs, RR1-RR7, solvent bottle, RS1, and wash-
solution bottles RS4 and RS5. The operation of the automated
synthesizer was then started. A solution of monoethyl malonate
◦
and 3-tritylthiopropanal (9) in DMF was stirred at 25 C under
a nitrogen atmosphere in the reaction vessel, RF1. A solution
of DMAP in DMF in the reagent reservoir, RR3, was added to
the reaction vessel, RF1. After stirring at 100 ◦C for 2 h, the
reaction was quenched by the addition of 1 M HCl from the
wash-solution bottle, RS4, at 25 ◦C. After the addition of EtOAc
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from the solvent bottle, RS1, the mixture was stirred and then
transferred to a centrifugal separator, SF. After centrifugation,
the two resulting phases were separated by measurement of their
electroconductivities with a sensor and were then transferred to
two receivers, SF1 and SF2. The aqueous phase in SF1 was taken
back to the reaction vessel, RF1. After the addition of EtOAc
from the solvent bottle, RS1, the mixture was stirred and then
transferred to the centrifugal separator, SF. After performing
the extraction procedure twice, the combined organic mixture
in the receiver, SF2, was washed with 1 M HCl, 5% aqueous
NaHCO3 and 10% aqueous NaCl from the wash-solution bottles,
RS6, RS5, and RS4, in the reaction flask, RF1. The organic
layer was separated in the centrifugal separator, SF, and was
transferred to the receiver, SF2. The organic layer in SF2 was
then passed through an anhydrous Na2SO4 plug, DT1, and was
collected in a round flask, CF1. Finally, the entire apparatus
was washed with water and acetone, and dried under reduced
pressure. The collected solution was purified manually using flash
silica gel column chromatography to give a mixture of ethyl (E)-5-
(tritylthio)pent-2-enoate and ethyl (E)-5-(tritylthio)pent-3-enoate
10 in an 88% combined yield.
Scheme 3 Synthesis of D-allo-isoleucine derived statine allyl ester 4.
Reagents and Conditions: a) carbonyldiimidazole, (EtO2CCH2CO2)2Mg,
THF, 76%; b) KBH4, MeOH, 85% (16a : 16b = 91 : 9); c) LiOH, THF–H2O,
68% (17a : 17b = 95 : 5); d) allyl bromide, K2CO3, DMF, 75%.
DIBAL reduction of the ethyl esters and subsequent Swern
oxidation were performed by using the automated synthesizer
to afford (E)-5-(tritylthio)pent-2-enal (12)19,26 as a single product
(Scheme 2).
The key step, asymmetric aldol reaction of enal 12,12 was carried
out with the use of the automated synthesizer (Scheme 2). n-BuLi
was manually added to a solution of (R)-N-acetyl-4-isopropyl-
5,5-diphenyl-2-oxazolidinone in THF at -78 ◦C under a nitrogen
atmosphere in the reaction vessel, RF1. After stirring for 1 h
at the same temperature, a solution of Cp2ZrCl2 in THF, in the
reagent reservoir, RR2, was added to the reaction mixture, and
Total synthesis of spiruchostatin B (2)
Prepared segments were coupled as shown in Scheme 4. In the
condensation of cysteine with the statine derivative 4, the order of
reagent addition greatly affected the racemization of cysteine. It
is well known that cysteine is highly susceptible to racemization,
especially after converting to the corresponding activated ester. In
◦
then warmed to -30 C. The resulting mixture was stirred at the
same temperature for 1 h and cooled to -50 ◦C. A solution of the
enal 12 in THF, in the reagent reservoir, RR3, was added to the
reaction mixture, and then warmed to 0 ◦C. After stirring for 1 h at
the same temperature, the reaction mixture in the reaction vessel,
RF1, was poured into 10% aqueous NH4Cl in another reaction
vessel, RF2. Automated workup and manual silica gel column
purification afforded a 90 : 10 mixture of aldols 13a and 13b in an
84% combined yield. The diastereomeric mixture was repurified
using silica gel column chromatography to give pure 13a.
Hydrolysis of 13a was performed manually. After simple
purification by silica gel column chromatography, the desired b-
hydroxy acid 719 was obtained in 92% yield (Scheme 2).
D-allo-Isoleucine derived statine allyl ester 4 was prepared
from N-Boc-D-allo-isoleucine (14) (Scheme 3). Condensation with
ethyl magnesium malonate,53 followed by stereoselective reduction
with KBH4 afforded the desired alcohol 16a as a major isomer.
Hydrolysis of the diastereomeric mixture of 16a and 16b, and
subsequent recrystallization afforded a white solid (17%). After
converting the obtained carboxylic acid into its corresponding allyl
ester, the ratio of 17a and 17b was determined by 1H NMR analysis
(17a : 17b = ca.50 : 50). On the other hand, the carboxylic acid
(68%) obtained in the filtrate was converted into its corresponding
benzyl ester, and the ratio of 17a and 17b was determined by HPLC
with a photodiode array (17a : 17b = 95 : 5). The desired product
17a obtained from the filtrate was treated with allyl bromide to
afford the allyl ester 4.
Scheme 4 Total synthesis of spiruchostatin B (2). Reagents and Con-
ditions: a) i) 4 M HCl; ii) 5, EDCI·HCl, HOBt, DIEA, 2 steps 92%);
b) i) Et2NH; ii) 6, EDCI·HCl, HOBt, DIEA, 2 steps 98%; c) i)
Et2NH; ii) 7, PyBOP, DIEA, 2 steps 76%; d) i) LiOH, THF–H2O;
ii) MNBA, DMAP,
2 steps 62%; f) I2, MeOH, 82%. EDCI =
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, HOBt = 1-hydroxyben-
zotriazole, DIEA = N,N-diisopropylethylamine, PyBOP = benzotria-
zole-1-yl-oxy-tripyrrolidinophosphonium hexafluorophosphate, MNBA =
2-methyl-6-nitrobenzoic anhydride.
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fact, racemization was observed by adding DIEA into the pre-
incubated solution of cysteine 5, hydrochloride salt of deprotected
4, EDCI·HCl, and HOBt. However, severe racemization (<7%)
was avoided by adding DIEA and EDCI·HCl into the premixed
solution of cysteine 5, HOBt, and the hydrochloride salt of
deprotected 4. Compound 3 was prepared from 18 by subsequent
condensation with D-alanine (6) and b-hydroxy acid 7 in good
yield. Unexpectedly, the allyl group of 3 was cleanly removed by
using conventional basic hydrolysis conditions to afford seco-acid
20. Macrolactonization of 20 using Shiina’s method54–57 followed
by disulfide bond formation, the same procedure used in our
total synthesis of spiruchostatin A,12 was performed to furnish
spiruchostatin B (2)18 in high yield. The spectral data of synthetic
2 were in good accordance with those of the natural product.6
Reporter gene assay in HEK 293 cell promoted cytomegalovirus
(CMV) was performed to evaluate the HDAC inhibitory activity
of the synthetic spiruchostatin A (1) and B (2).58 ED50 values of
both 1 and 2 were determined to be 200 nM. Almost the same
level of HDAC inhibitory activity was observed in the synthetic
spiruchostatins.
by distillation from CaH2. MeOH was dried by distillation from
magnesium contained with a catalytic amount of iodine.
Ethyl (E)-5-(tritylthio)-2-pentenoate and ethyl
(E)-5-(tritylthio)-3-pent-3-enoate (10)
A solution of tritylthiol (10.0 g, 36.2 mmol, 8.3 equiv.) in CH2Cl2
(80 mL) in RF1 was added Et3N (7.56 mL, 54.3 mmol, 12.5 equiv.,
RR1) and acrolein (8) (3.44 mL, 4.36 mmol, 1.0 equiv., RR2). After
being stirred at room temperature for 1 h, the reaction mixture
was transferred to a round flask CF1. After removal of solvent,
the crude 3-tritylthiopropanal (9) was used in the next reaction
without further purification.
Solutions of monoethyl malonate (8.13 g, 61.5 mmol, 1.7
equiv., RR1) in DMF (15 mL, RR1) and 3-tritylthiopropanal (9)
(12.0 g, 36.2 mmol, 1.0 equiv., RR2) in DMF (60 mL, RR2) were
transferred into RF1. The resulting mixture was added DMAP
(31.0 mg, 0.254 mmol, 0.007 equiv., RR3) in DMF (8.0 mL, RR3)
at room temperature. After being stirred at 100 ◦C for 2 h, the
reaction mixture was quenched by addition of 1 M HCl (60 mL,
RS4) and diluted with EtOAc (60 mL, RS1) and transferred to
SF. After centrifugation, two phases were separated. The separated
aqueous phase in SF1 was taken back to RF1 and extracted with
EtOAc (60 mL, RS1). The mixture was transferred to SF and
centrifuged, and the two phases were separated. After repeating
this extraction process twice, the combined organic phase in
SF2 was transferred to RF1. The organic phase was washed
with 1 M HCl (60 mL, RS4), 5% aqueous NaHCO3 solution
(60 mL, RS5) and 10% aqueous NaCl solution (60 mL, RS6).
The resulting organic phase in SF2 was dried by passing through
anhydrous Na2SO4 (DT1) and transferred to a round flask CF1.
After removal of solvent, the residue was purified by column
chromatography on silica gel (10% EtOAc in hexane) to give a
mixture of ethyl (E)-5-(tritylthio)-2-pentenoate and ethyl (E)-5-
(tritylthio)-3-pentenoate 10 (12.8 g, 31.8 mmol, 88%) as a white
solid. Ethyl (E)-5-(tritylthio)-2-pentenoate: 1H NMR (270 MHz,
CDCl3): d 7.42–7.19 (m, 15H), 6.76 (dt, J = 15.5, 6.8 Hz, 1H), 5.69
(d, J = 16.0 Hz, 1H), 4.15 (q, J = 6.8 Hz, 2H), 2.28 (dd, J = 8.2,
6.8 Hz, 2H), 2.17 (dt, J = 7.3, 6.8 Hz, 2H), 1.26 (t, J = 6.8 Hz,
Conclusions
In summary, HDAC inhibitor spiruchostatin B (2) was successfully
synthesized. b-Hydroxy acid 7, the common synthetic interme-
diate for cysteine-containing natural product HDAC inhibitors
such as spiruchostatins, FK228, FR901375 and largazole was
efficiently prepared. A total of 5 steps were successfully performed
using an automated synthesizer that was developed by our group
-including an asymmetric aldol reaction that requires careful
synthetic manipulation to obtain a high yield and a high stereose-
lectivity. Prepared segments 4–7 were coupled in good yield while
suppressing the undesired racemization of a cysteine segment.
Shiina’s method was employed to form a macrolactone ring
and subsequent disulfide formation afforded the desired product.
The developed method should be very useful for combinatorial
synthesis of cysteine-containing natural product HDAC inhibitor
analogues.
1
3H). Ethyl (E)-5-(tritylthio)-3-pentenoate: H NMR (270 MHz,
CDCl3): d 7.42–7.19 (m, 15H), 5.57 (dt, J = 15.4, 6.8 Hz, 1H), 5.44
(dt, J = 15.5, 7.3 Hz, 1H), 4.10 (q, J = 7.2 Hz, 2H), 2.96 (d, J =
6.8 Hz, 2H), 2.80 (d, J = 7.2 Hz, 2H), 1.24 (t, J = 6.8 Hz, 3H).
Experimental section
General
NMR spectra were recorded in the indicated solvents. Chemical
shifts are reported in unit of parts per million (ppm) from
tetramethylsilane with the solvent resonance as the internal
(E)-5-(Tritylthio)-2-penten-1-ol and
(E)-5-(tritylthio)-3-penten-1-ol (11)
1
standard (CHCl3: d 7.26 ppm for H, CDCl3: d 77.0 ppm for
To a solution of ethyl (E)-5-(tritylthio)-2-pentenoate and ethyl
(E)-5-(tritylthio)-3-pentenoate 10 (4.77 g, 11.8 mmol, 1.0 eq.) in
CH2Cl2 (35 mL) in RF1 was added DIBAL (0.99 M in toluene,
36.0 mL, 35.5 mmol, 3.0 equiv.) dropwise at -20 ◦C and allowed to
warm to room temperature. After being stirred for 2 h, the reaction
mixture was quenched by addition of Rochelle salt (30 mL, RR7)
and stirred for 30 min. The reaction mixture was diluted with 1 M
HCl (20 mL, RS6) and EtOAc (100 mL, RS1) and transferred to
SF. After centrifugation, two phases were separated. The separated
aqueous phase in SF1 was taken back to RF1 and extracted with
EtOAc (100 mL, RS1). The mixture was transferred to SF and
centrifuged, and the two phases were separated. After repeating
13C). Data are reported as follows: chemical shift, multiplicity
(s; singlet, d; doublet, t; triplet, q; quartet, m; multiplet, br;
broad), coupling constants (Hz) and integration. All reactions
were monitored by thin-layer chromatography using E. Merck
silica gel plates (60F-254) pre-coated plates (0.25 mm). TLC
visualization was done with UV light and/or 5% ethanolic p-
anisaldehyde or 10% ethanolic phosphomolybdic acid followed
by heating. Flash column chromatography was performed on
Silica Gel 60 N, purchased from Kanto Chemical Co. THF was
dried by distillation from sodium/benzophenone ketyl. CH2Cl2
was dried by distillation from P2O5. CH3CN and DMF were dried
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this extraction process twice, the combined organic phase in SF2
was transferred to RF1. The organic phase was washed with 1 M
HCl (80 mL, RS6), 5% aqueous NaHCO3 solution (80 mL, RS5)
and 10% aqueous NaCl solution (80 mL, RS4). The resulting
organic phase in SF2 was dried by passing through anhydrous
Na2SO4 (DT1) and transferred to a round flask CF1. After removal
of solvent, the residue was purified by column chromatography
on silica gel (20% EtOAc in hexane) to give a mixture of (E)-5-
(tritylthio)-2-penten-1-ol19 and (E)-5-(tritylthio)-3-penten-1-ol 11
(3.21 g, 8.90 mmol, 75%) as a colorless oil. (E)-5-(Tritylthio)-2-
penten-1-ol: 1H NMR (270 MHz, CDCl3): d 7.42–7.18 (m, 15H),
5.55–5.43 (m, 2H), 4.03 (d, J = 3.9 Hz, 2H), 2.22 (m, 2H), 2.09
2.6 equiv.) at -78 ◦C by attaching EtOH-dry ice bath manually
to RF1. After being stirred for 1 h at the same temperature,
Cp2ZrCl2 (1.07 g, 3.66 mmol, 2.7 equiv., RR2) in THF (14 mL)
was added to RF1 and the reaction mixture was allowed to warm
to -30 ◦C. After being stirred for 1 h, the reaction mixture was
cooled to -50 ◦C and added a solution of (E)-5-(tritylthio)-2-
pentenal (12) (486 mg, 1.35 mmol, 1.0 equiv., RR3) in THF (10
mL) at the same temperature and allowed to warm to 0 ◦C. After
being stirred for 1 h at the same temperature, the reaction mixture
was transferred to RF2 to be poured into 10% aqueous NH4Cl
solution (40 mL) placed in RF2 and diluted with EtOAc (40 mL,
RS1) and transferred to SF. After centrifugation, two phases
were separated. The separated aqueous phase in SF1 was taken
back to RF1 and extracted with EtOAc (60 mL, RS1). The
mixture was transferred to SF and centrifuged, and the two phases
were separated. After repeating this extraction process twice, the
combined organic phase in SF2 was transferred to RF1. The
organic phase was washed with 5% aqueous NaHCO3 solution
(100 mL, RS4) and 10% aqueous NaCl solution (100 mL, RS6).
The resulting organic phase in SF2 was dried by passing through
anhydrous Na2SO4 (DT1) and transferred to a round flask CF1.
After removal of solvent, the residue was purified by column
chromatography on silica gel (20% EtOAc in hexane) to give
a mixture of (E)-(S)-3-hydroxy-1-[(R)-4-isopropyl-5,5-diphenyl-
2-oxazolidinone-3-yl]-7-tritylthio-4-hepten-1-one (13a) and its di-
astereomer 13b [771 mg, 1.13 mmol, 84%, 13a:13b = 90 : 10 (the
ratio was determined by HPLC with photodiode array)] as a
colorless oil. The diastereomeric mixture was purified by column
chromatography on silica gel (1% EtOAc in toluene) to give pure
13a (441 mg, 0.647 mmol, isolated yield was 48%) as a colorless
oil. 1H NMR (400 MHz, CDCl3): d 7.47–7.15 (m, 25H), 5.49 (dt,
J = 15.0, 6.8 Hz, 1H), 5.37 (m, 2H), 4.47 (m, 1H), 3.15 (dd, J =
16.9, 2.9 Hz, 1H), 2.82 (dd, J = 16.9, 8.2 Hz, 1H), 2.17 (dd, J =
7.7, 7.3 Hz, 2H) 2.00 (m, 3H), 0.87 (d, J = 6.8 Hz, 3H), 0.74 (d, J =
6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 171.8, 152.9, 144.9,
142.0, 137.9, 131.7, 130.1, 129.5, 128.9, 128.7, 128.4, 128.0, 127.8,
126.5, 125.8, 125.4, 89.6, 68.5, 66.5, 64.5, 42.2, 31.3, 29.9, 21.7,
16.3; IR (neat): 3509, 3060, 3030, 2965, 2927, 2853, 1785, 1699,
1596, 1493, 1466, 1449, 1367, 1319, 1211, 1176, 1094, 1035, 1002,
986, 845, 701, 667, 617, 506 cm-1; [a]D26 = +85 (c 0.81, CHCl3).
HRMS (ESI-TOF): calcd for [C45H45NO3S+H]+ 680.3198, found
680.3168.
1
(m, 2H). (E)-5-(Tritylthio)-3-penten-1-ol: H NMR (270 MHz,
CDCl3): d 7.42-7.18 (m, 15H), 5.55–5.43 (m, 2H), 3.58 (t, J =
6.3 Hz, 2H), 2.79 (d, J = 4.9 Hz, 2H), 2.22 (m, 2H).
(E)-5-(Tritylthio)-2-pentenal (12)
Oxalyl chloride (796 mL, 9.28 mmol, 1.5 equiv.) was placed in RF1
◦
and cooled to -78 C by attaching EtOH-dry ice bath manually
to RF1. A solution of DMSO (1.32 mL, 18.6 mmol, 3.0 equiv.,
RR1) in CH2Cl2 (27 mL) was added to RF1 at -78 ◦C. After
being stirred at the same temperature for 60 min, a mixture of (E)-
5-(tritylthio)-2-penten-1-ol and (E)-5-(tritylthio)-3-penten-1-ol 11
(2.23 g, 6.19 mmol, 1.0 equ◦ iv., RR2) in CH2Cl2 (10 mL) was added
dropwise to RF1 at -78 C and stirred at the same temperature
for an additional 30 min. Et3N (2.58 mL, 18.6 mmol, 3.0 equiv.,
RR3) in CH2Cl2 (3.0 mL) was added to RF1 at -78 ◦C and
allowed to warm to 0 ◦C. After being stirred for 1 h, the reaction
mixture was quenched by addition of 1 M HCl (60 mL, RS6) and
diluted with EtOAc (80 mL, RS1) and transferred to SF. After
centrifugation, two phases were separated. The separated aqueous
phase in SF1 was taken back to RF1 and extracted with EtOAc
(60 mL, RS1). The mixture was transferred to SF and centrifuged,
and the two phases were separated. After repeating this extraction
process twice, the combined organic phase in SF2 was transferred
to RF1. The organic phase was washed with 5% aqueous NaHCO3
solution (40 mL, RS5) and 10% aqueous NaCl solution (40 mL,
RS4). The resulting organic phase in SF2 was dried by passing
through anhydrous Na2SO4 (DT1) and transferred to a round
flask CF1. After removal of solvent, the residue was purified by
column chromatography on silica gel (8% EtOAc in hexane) to
give (E)-5-(tritylthio)pent-2-enal (12)19,26 (1.23 g, 3.44 mmol, 56%)
1
as a white solid. H NMR (270 MHz, CDCl3): d 9.43 (d, J =
(E)-(S)-3-Hydroxy-7-tritylthio-4-heptenoic acid (7)
7.7 Hz, 1H), 7.43–7.20 (m, 15H), 6.63 (dt, J = 15.4, 6.3 Hz, 1H),
5.98 (dd, J = 15.4, 7.7 Hz, 1H), 2.37–2.29 (m, 4H); 13C NMR
(67.8 MHz, CDCl3): d 193.6, 155.6, 144.5, 133.6, 129.5, 127.9,
126.7, 67.0, 31.7, 30.0; IR (solid): 3062, 2931, 2830, 2755, 1964,
1686, 1634, 1592, 1486, 1440, 1396, 1317, 1246, 1208, 1181, 1154,
1112, 1081, 1035, 984, 959, 886, 840, 814, 763, 741, 699, 672, 628,
617, 571, 519, 507, 477, 458 cm-1. HRMS (ESI-TOF): calcd for
[C24H22O+NH4]+ 376.1735, found 376.1751.
To
a
solution of (E)-(S)-3-hydroxy-1-[(R)-4-isopropyl-5,5-
diphenyl-2-oxazolidinone-3-yl]-7-tritylthio-4-hepten-1-one (13a)
(320 mg, 0.470 mmol, 1.0 equiv.) in THF (3.0 mL) and MeOH
(3.0 mL) was added 1 M aqueous sodium hydroxide (100 mL,
1.00 mmol, 2.1 equiv.) at 0 ◦C. After being stirred at the same
temperature for 1 h, the reaction mixture was concentrated in
vacuo. The res◦idue was diluted with EtOAc and quenched with
3 M HCl at 0 C. The aqueous layer was extracted with EtOAc.
The combined organic layers were washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (1.5% MeOH in CHCl3) to
give (E)-(S)-3-hydroxy-7-tritylthio-4-heptenoic acid (7)19 (171 mg,
0.431 mmol, 92%) as a white solid. 1H NMR (400 MHz, CDCl3): d
7.51–7.19 (m, 15H), 5.59 (ddd, J = 15.5, 6.8, 6.3 Hz, 1H), 5.42 (dd,
(E)-(S)-3-Hydroxy-1-[(R)-4-isopropyl-5,5-diphenyl-2-
oxazolidinone-3-yl]-7-tritylthio-4-hepten-1-one (13a)
(R)-N-Acetyl-4-isopropyl-5,5-diphenyl-2-oxazolidinone (1.96 g,
3.39 mmol, 2.5 equiv.) placed in RF1 was added THF (12 mL)
and added n-BuLi (1.59 M in hexane, 2.21 mL, 3.52 mmol,
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J = 15.5, 6.3 Hz, 1H), 4.45 (m, 1H), 2.54 (m, 2H), 2.22 (m, 2H),
2.09 (m, 2H); 13C NMR (100 MHz, CDCl3): d 176.5, 144.9, 131.6,
130.7, 129.6, 127.9, 126.6, 68.5, 66.6, 41.1, 31.4, 31.3; IR (neat):
3417, 3056, 3030, 2925, 1713, 1595, 1489, 1444, 1280, 1183, 1101,
1083, 1034, 1002, 972, 743, 700, 676, 621, 506, cm-1; [a]D25 = -5.0
(c 1.6, CH2Cl2); HRMS (ESI-TOF): calcd for [C26H26O3S+Na]+
441.1500, found 441.1495.
(c 0.57, MeOH); HRMS (ESI-TOF): calcd for [C15H31NO5+H]+
304.2124, found 304.2124.
(3S,4R,5S)-4-(t-Butoxycarbonylamino)-3-hydroxy-5-
methylheptanoic acid (17a)
To a mixture of 16a and 16b (1.88 g, 6.20 mmol, 1.0 equiv.) in THF
(15 mL) was added 1 M aqueous LiOH (15.0 mL, 15.0 mmol, 2.4
equiv.) at 0 ◦C. After being stirred at room temperature for 1 h, the
reaction mixture was quenched with 1 M HCl at 0 ◦C and the aque-
ous layer was extracted with EtOAc. The combined organic layers
were washed with brine, dried over Na2SO4, and concentrated in
vacuo. The residue was recrystallized from EtOAc–hexane to give
a mixture of (3S,4R,5S)-4-(t-butoxycarbonylamino)-3-hydroxy-5-
methylheptanoic acid (17a)61,62 and its diastereomer 17b [1.05 g,
1.05 mmol, 17%, 17a : 17b = 50 : 50, (The mixture of 17a : 17b was
converted to corresponding allyl esters by treating with K2CO3,
allyl bromide in DMF. The ratio of 17a and 17b was determined by
1H NMR analysis) as a white solid. The filtrate was concentrated
to afford desired 17a as a major component [colorless oil, 1.16 g,
4.20 mmol, 68%, 17a:17b = 95 : 5 (The mixture of 17a and 17b was
converted to corresponding benzyl esters by treating with K2CO3,
benzyl bromide in DMF. The ratio of 17a and 17b was determined
Ethyl (4R,5S)-4-(t-butoxycarbonylamino)-3-oxo-
5-methylheptanoate (15)
To a solution of N-Boc-D-allo-isoleucine (14) (3.56 g, 15.4 mmol,
1.0 equiv.) in THF (45 mL) was added carbonyldiimidazole (4.25 g,
26.2 mmol, 1.7 equiv.). After being stirred at room temperature
for 1 h, magnesium ethyl malonate (7.50 g, 26.2 mmol, 1.7 equiv.)
was added and the resulting mixture was stirred for 27 h. The
reaction mixture was poured into saturated aqueous NH4Cl and
the aqueous layer was extracted with EtOAc. The combined
organic layers were washed with saturated aqueous NaHCO3,
brine, dried over Na2SO4, and concentrated in vacuo. The residue
was purified by column chromatography on silica gel (10% EtOAc
in hexane) to give ethyl (4R,5S)-4-(t-butoxycarbonylamino)-3-
oxo-5-methylheptanoate (15)59 (3.54 g, 11.8 mmol, 76%) as a
colorless oil. 1H NMR (270 MHz, CDCl3): d 5.04 (d, J = 8.9 Hz,
1H), 4.41 (m, 1H), 4.12 (q, J = 7.3 Hz, 2H), 3.46 (s, 2H), 1.92 (m,
1H), 1.37 (s, 9H), 1.21 (t, J = 7.3 Hz, 3H), 0.95–0.77 (m, 2H), 0.90
(t, J = 7.3 Hz, 3H), 0.71 (d, J = 6.9 Hz, 3H); 13C NMR (67.5 MHz,
CDCl3): d 202.7, 167.0, 156.1, 80.1, 62.9, 61.7, 47.1, 36.2, 28.5,
27.0, 14.3, 14.1, 12.1; IR (neat): 3370, 2972, 2936, 2879, 1749,
1712, 1657, 1504, 1459, 1391, 1367, 1316, 1246, 1167, 1097, 1032,
779 cm-1; [a]D21 = -30 (c 1.2, CHCl3); HRMS (ESI-TOF): calcd for
[C15H28NO5+H]+ 302.1967, found 302.1962.
1
by HPLC with photodiode array)]. 17a: H NMR (400 MHz,
CDCl3) d 4.49 (d, J = 9.7 Hz, 1H), 3.95 (m, 1H), 3.62 (m, 1H),
2.69–2.35 (m, 2H), 1.88 (m, 1H), 1.44 (s, 9H), 1.41–1.22 (m, 2H),
0.93 (t, J = 7.3 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H). [a]2D1 = -6.5
(c 0.75, CHCl3); HRMS (ESI-TOF): calcd for [C13H26NO5+H]+
276.1811, found 276.1814.
Allyl (3S,4R,5S)-4-(t-butoxycarbonylamino)-3-hydroxy-5-
methylheptanoate (4)
To a mixture of 17a and 17b (1.16 g, 4.20 mmol, 1.0 equiv.) in DMF
(20 mL) was added K2CO3 (695 mg, 5.03 mmol, 1.2 equiv.) and
allyl bromide (532 mL, 6.29 mmol, 1.5 equiv.). After being stirred
at room temperature for 1.5 h, the reaction mixture was poured
Ethyl (3S,4R,5S)-4-(t-butoxycarbonylamino)-3-hydroxy-5-
methylheptanoate (16a)
◦
into 1 M HCl at 0 C and the aqueous layer was extracted with
To a solution of b-keto ester 15 (2.21 g, 7.34 mmol, 1.0 equiv.) in
MeOH (40 mL) was added KBH4 (1.98 g, 36.7 mmol, 5.0 equiv.)
at -78 ◦C. The reaction mixture was allowed to warm to 0 ◦C
slowly and stirred at the same temperature for 1 h. The reaction
mixture was quenched with AcOH at 0 ◦C and concentrated in
vacuo. The residue was diluted with EtOAc and water. The aqueous
layer was extracted with EtOAc. The combined organic layers
were washed with saturated aqueous NaHCO3, brine, dried over
Na2SO4, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (20% EtOAc in hexane) to
give a mixture of ethyl (3S,4R,5S)-4-(t-butoxycarbonylamino)-3-
hydroxy-5-methylheptanoate 16a59,60 and its diastereomer 16b59,60
[1.88 g, 6.20 mmol, 85%, 16a:16b = 91 : 9 (the ratio was determined
by 1H NMR peak area)] as a colorless oil. 16a: 1H NMR (400 MHz,
CDCl3): d 4.42 (d, J = 10.1 Hz, 1H), 4.18 (q, J = 7.3 Hz, 2H), 3.90
(m, 1H), 3.64 (m, 1H), 3.25 (d, J = 4.4 Hz, 1H), 2.63 (dt, J = 16.5,
2.5 Hz, 1H), 2.51 (dd, J = 16.5, 8.9 Hz, 1H), 1.93 (m, 1H), 1.43 (s,
9H), 1.38–1.20 (m, 2H), 1.23 (t, J = 7.3 Hz, 3H), 0.92 (t, J = 7.3 Hz,
3H), 0.86 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): d
173.1, 156.0, 79.1, 68.9, 60.5, 56.6, 38.7, 33.8, 28.2, 26.9, 13.9, 13.0,
11.5; IR (neat): 3451, 3372, 2968, 2935, 2878, 1716, 1701, 1522,
1457, 1391, 1367, 1251, 1171, 1072, 989, 864, 775 cm-1; [a]D24 = -5.2
EtOAc. The combined organic layers were washed with saturated
aqueous NaHCO3, brine, dried over Na2SO4, and concentrated
in vacuo. The residue was purified by column chromatography
on silica gel (15% EtOAc in hexane) to give allyl (3S,4R,5S)-4-(t-
butoxycarbonylamino)-3-hydroxy-5-methylheptanoate (4) (1.45 g,
4.60 mmol, quant.) as a colorless oil. 1H NMR (270 MHz, CDCl3)
d 5.87 (m, 1H), 5.28 (d, J = 17.9 Hz, 1H), 5.20 (d, J = 10.2 Hz,
1H), 4.62 (d, J = 5.6 Hz, 2H), 4.41 (d, J = 9.9 Hz, 1H), 3.91 (m,
1H), 3.65 (m, 1H), 2.69–2.47 (m, 2H), 1.91 (m, 1H), 1.44 (s, 9H),
1.36–1.22 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H);
13C NMR (67.5 MHz, CDCl3): d 173.0, 156.2, 131.9, 118.6, 79.5,
69.2, 65.5, 56.7, 38.7, 34.0, 28.4, 27.1, 13.2, 11.7; IR (neat): 3450,
3374, 2967, 1717, 1699, 1512, 1457, 1392, 1367, 1251, 1172, 1074,
989, 931, 584 cm-1; [a]D26 = -6.4 (c 2.9, CHCl3); HRMS (ESI-TOF):
calcd for [C16H30NO5+H]+ 316.2124, found 316.2126.
Allyl (3S,4R,5S)-4-[(S)-2-(9H-fluoren-9-ylmethoxycarbonyl-
amino)-3-tritylthiopropionylamino]-3-hydroxy-5-methylheptanoate
(18)
D-allo-Isoleucine derived statine allyl ester 4 (447 mg, 1.42 mmol,
1.0 equiv.) was treated with HCl (4 M in dioxane, 10 mL) at
3830 | Org. Biomol. Chem., 2011, 9, 3825–3833
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0 ◦C. After being stirred at room temperature for 2 h, the reaction
mixture was concentrated in vacuo. The crude amine was dissolved
in DMF (10 mL) and added Fmoc-D-Cys(Trt)-OH (5) (912 mg,
1.56 mmol, 1.1 equiv.), HOBt (287 mg, 2.12 mmol, 1.5 equiv.),
DIEA (739 mL, 4.25 mmol, 3.0 equiv.) and EDCI·HCl (408 mg,
2.12 mmol, 1.5 equiv.) at 0 ◦C. After being stirred at room
temperature for 2 h, the reaction mixture was quenched with
saturated aqueous NH4Cl and the aqueous layer was extracted
with EtOAc. The combined organic layers were washed with
1 M HCl, saturated aqueous NaHCO3, brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (30% EtOAc in hexane) to give al-
lyl (3S,4R,5S)-4-[(S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-
3-tritylthiopropionylamino]-3-hydroxy-5-methylheptanoate (18)
(1.02 g, 1.30 mmol, 2 steps 92%) as a colorless oil and a small
amount (<7%) of C2 (cysteine) epimer of 18. 1H NMR (270 MHz,
CDCl3): d 7.74 (m, 2H), 7.54 (t, J = 6.9 Hz, 2H), 7.43–7.17 (m,
19H), 5.83 (m, 2H), 5.28 (d, J = 16.8 Hz, 1H), 5.20 (d, J = 10.6 Hz,
1H), 4.89 (d, J = 6.9 Hz, 1H), 4.55 (d, J = 4.6 Hz, 2H), 4.40 (m,
2H), 4.20–3.64 (m, 3H), 4.17 (m, 1H), 2.66–2.43 (m, 4H), 1.89
(m, 1H), 1.22–1.06 (m, 2H), 0.85 (t, J = 7.9 Hz, 3H), 0.80 (d, J =
6.6 Hz, 3H); 13C NMR (67.5 MHz, CDCl3): d 173.1, 170.4, 156.1,
144.4, 143.6, 141.4, 131.8, 129.6, 128.2, 127.9, 127.2, 127.0, 125.0,
120.1, 118.7, 68.6, 67.5, 67.1, 65.5, 55.1, 54.3, 47.1, 38.4, 33.7,
33.2, 27.1, 13.3, 11.7; IR (neat): 3316, 2961, 1716, 1665, 1527,
1448, 1248, 1179, 1034, 742, 701 cm-1; [a]D25 = +1.6 (c 1.1, CHCl3).
HRMS (ESI-TOF): calcd for [C48H50N2O6S+H]+ 783.3468, found
783.3467.
1530, 1448, 1220, 758, 742, 701 cm-1; [a]D26 = +2.6 (c 3.1, CHCl3);
HRMS (ESI-TOF): calcd for [C51H56N3O7S+H]+ 854.3839, found
854.3837.
Allyl (3S,4R,5S)-4-[(S)-2-{(R)-2-[(E)-(S)-3-hydroxy-7-trithylthio-
4-heptenoylamino]propionylamino}-3-tritylthiopropionylamino]-3-
hydroxy-5-methylheptanoate (3)
To a solution of N-Fmoc alanine derivative 19 (191 mg,
0.224 mmol, 1.0 equiv.) in CH3CN (5 mL) was added Et2NH
(1 mL). After being stirred at room temperature for 1 h, the
reaction mixture was concentrated in vacuo. The residue was
azeotroped with toluene and hexane twice, then dissolved in
CH2Cl2 (5 mL) and CH3CN (5 mL). To this solution was added
(E)-(S)-3-hydroxy-7-tritylthio-4-heptenoic acid (7) (85.6 mg,
0.216 mmol, 1.0 equiv.), DIEA (113 mL, 0.648 mmol, 3.0 equiv.)
and PyBOP (170 mg, 0.324 mmol, 1.5 equiv.) and stirred at
room temperature for 2 h. The reaction mixture was quenched
with saturated aqueous NH4Cl and the aqueous layer was
extracted with EtOAc. The combined organic layers were washed
with brine, dried over Na2SO4, and concentrated in vacuo. The
residue was purified by column chromatography on silica gel (1%
MeOH in CHCl3) to give allyl (3S,4R,5S)-4-[(S)-2-{(R)-2-[(E)-
(S)-3-hydroxy-7-trithylthio-4-heptenoylamino]propionylamino}-
3-tritylthiopropionylamino]-3-hydroxy-5-methylheptanoate (3)
(169.0 mg, 0.164 mmol, 2 steps 76%) as a colorless oil. 1H NMR
(400 MHz, CDCl3): d 7.43–7.17 (m, 30H), 6.94 (d, J = 7.2 Hz,
1H), 6.09 (m, 2H), 5.85 (m, 1H), 5.45 (m, 1H), 5.34–5.18 (m, 3H),
4.55 (d, J = 10.6 Hz, 2H), 4.34–4.25 (m, 2H), 4.03–3.93 (m, 3H),
2.74–2.41 (m, 4H), 2.36–2.05 (m, 7H), 1.89 (m, 1H), 1.33 (d, J =
7.2 Hz, 3H), 1.15–1.08 (m, 2H), 0.97–0.80 (m, 6H); 13C NMR
(67.5 MHz, CDCl3): d 173.1, 172.2, 171.8, 170.4, 144.9, 144.3,
132.4, 132.0, 130.4, 129.5, 129.1, 128.2, 128.0, 127.1, 126.7, 118.4,
69.9, 68.6, 67.0, 66.7, 65.4, 55.7, 52.8, 51.0, 44.0, 38.4, 34.1, 33.1,
31.3, 29.8, 27.1, 17.5, 13.7, 11.8; IR (neat): 3273, 1736, 1624,
1528, 1444, 1219, 1168, 744, 699. [a]2D3 = -4.6 (c 1.1, CHCl3).
Allyl (3S,4R,5S)-4-{(S)-2-[(R)-2-(9H-fluoren-9-ylmethoxycarbo-
nylamino)propionylamino]-3-tritylthiopropionylamino}-3-hydroxy-
5-methylheptanoate (19)
N-Fmoc cysteine derivative 18 (1.01 g, 1.29 mmol, 1.0 equiv.)
in CH2Cl2 (5.0 mL) was added Et2NH (5.0 mL). After being
stirred at room temperature for 2 h, the reaction mixture was
concentrated in vacuo. The residue was azeotroped with toluene
and hexane twice, then dissolved in DMF (10 mL). To this
solution was added N-Fmoc-D-Ala-OH (6) (441 mg, 1.42 mmol,
1.1 equiv.), HOBt (262 mg, 1.94 mmol, 1.5 equiv.) and EDCI·HCl
(373 mg, 1.94 mmol, 1.5 equiv.) at room temperature. 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 EtOAc. The combined organic layers were
washed with 1 M HCl, saturated aqueous NaHCO3, brine, dried
over Na2SO4, and concentrated in vacuo. The residue was purified
by column chromatography on silica gel (45% EtOAc in hexane)
to give allyl (3S,4R,5S)-4-{(S)-2-[(R)-2-(9H-fluoren-9-ylmethoxy-
carbonylamino)propionylamino]-3-tritylthiopropionylamino}-3-
hydroxy-5-methylheptanoate (19) (1.09 g, 1.27 mmol, 2 steps 98%)
(2S,6R,9S,12R,13S)-12-(S)-sec-Butyl-13-hydroxy-6-methyl-2-
[(E)-4-tritylthiomethyl-2-butenyl]-9-tritylthiomethyl-1-oxa-5,8,11-
triaza-cyclopentadecane-4,7,11,15-tetraone (20)
To a solution of seco-acid derivative 3 (49.0 mg, 47.0 mmol, 1.0
equiv.) in THF (2.5 mL) was added water (700 mL) and 1 M
aqueous LiOH (150 mL, 150 mmol, 3.2 equiv.) at 0 ◦C. After being
stirred at the same temperature for 1.5 h, the reaction mixture
◦
was quenched with 1 M HCl at 0 C and the aqueous layer was
extracted with EtOAc. 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.
To a solution of MNBA (32.4 mg, 94.1 mmol, 2.0 equiv.)
and DMAP (23.0 mg, 188 mmol, 4.0 equiv.) in CH2Cl2 (30 mL)
was added dropwise crude acid in CH2Cl2 (20 mL) over 24 h
and stirred at room temperature for further 24 h. The reaction
mixture was concentrated in vacuo. The residue was purified by
column chromatography on silica gel (1% MeOH in CHCl3) to
give (2S,6R,9S,12R,13S)-12-(S)-sec-butyl-13-hydroxy-6-methyl-
2-[(E)-4-tritylthiomethyl-2-butenyl]-9-tritylthiomethyl-1-oxa-5,8,
11-triaza-cyclopentadecane-4,7,11,15-tetraone (20) (28.5 mg, 293
mmol, 2 steps 62%) as a white solid. 1H NMR (400 MHz, CDCl3):
1
as a colorless oil. H NMR (270 MHz, CDCl3): d 7.75 (d, J =
7.6 Hz, 2H), 7.55 (m, 2H), 7.47–7.12 (m, 19H), 6.45 (d, J = 6.9 Hz,
1H), 6.19 (d, J = 7.6 Hz, 1H), 5.82 (m, 1H), 5.30–5.15 (m, 3H), 4.54
(d, J = 4.6 Hz, 2H), 4.38 (m, 2H), 4.20–3.98 (m, 5H), 2.90–2.39 (m,
4H), 1.92 (m, 1H), 1.27 (m, 3H), 1.23–1.10 (m, 2H), 1.08–0.78 (m,
6H); 13C NMR (67.5 MHz, CDCl3): d 172.9, 172.2, 170.1, 156.0,
144.3, 143.8, 143.5, 141.2, 131.9, 129.4, 128.0, 127.7, 127.1, 126.8,
124.9, 120.0, 118.2, 68.4, 67.0, 65.3, 55.6, 52.5, 50.7, 47.0, 38.6,
33.9, 32.9, 27.0, 18.6, 13.3, 11.7; IR (neat): 3306, 2964, 1709, 1647,
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d 7.42–7.19 (m, 30H), 6.83 (d, J = 7.9 Hz, 1H), 6.71 (brs, 1H),
5.86 (brs, 1H), 5.66–5.54 (m, 2H), 5.32 (dd, J = 15.5, 6.6 Hz, 1H),
4.25–4.18 (m, 2H), 3.79 (m, 1H), 3.17 (m, 1H), 2.65–2.37 (m, 6H),
2.17 (t, J = 7.3 Hz, 2H), 2.05–1.83 (m, 3H), 1.34 (d, J = 6.9 Hz,
3H), 1.17–1.11 (m, 2H), 0.92–0.78 (m, 6H); IR (solid): 3294, 3059,
2960, 2926, 1734, 1661, 1542, 1444, 1378, 1252, 1179, 742, 700,
622 cm-1. [a]2D7 = -23 (c 0.095, CHCl3).
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(-)-Spiruchostatin B (2)
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To a solution of iodine (15.1 mg, 59.5 mmol, 10 equiv.) in CH2Cl2 (9
mL) and MeOH (1 mL) was dropwisely added a solution of trityl
thiol 20 (5.8 mg, 6.0 mmol, 1.0 equiv.) in CH2Cl2 (13.5 mL) and
MeOH (1.5 mL) at room temperature. After being stirred at room
temperature for 3 h, the reaction mixture was quenched with 10%
aqueous Na2S2O3 and saturated aqueous NaHCO3. The aqueous
layer was extracted with CHCl3. The combined organic layers
were dried over Na2SO4 and concentrated in vacuo. The residue
was purified by column chromatography on silica gel (5% MeOH
in CHCl3) to give spiruchostatin B (2) (2.4 mg, 4.9 mmol, 82%)
as a white solid. The spectral data of 2 were in good accordance
with reported values.6,18 1H NMR (400 MHz, CDCl3): d 7.29 (d,
J = 10.6 Hz, 1H), 6.69 (d, J = 9.7 Hz, 1H), 6.46 (m, 1H), 5.82
(brs, 1H), 5.64 (d, J = 15.5 Hz, 1H), 5.50 (brs, 1H), 4.95 (dt, J =
8.7, 3.9 Hz, 1H), 4.66 (m, 1H), 4.21 (dq, J = 3.9, 7.2 Hz, 1H),
3.42 (m, 1H), 3.37 (dd, J = 7.0, 13.1 Hz, 1H), 3.20–3.09 (m, 2H),
2.95–2.88 (m, 1H), 2.85–2.72 (m, 3H), 2.73 (d, J = 3.9 Hz, 2H)
2.57 (d, J = 13.1 Hz, 1H), 2.47 (m, 1H), 2.09 (m, 1H), 1.52 (d,
J = 7.2 Hz, 3H), 1.26 (m, 2H), 0.93 (d, J = 6.8 Hz, 3H), 0.92 (t,
J = 6.8 Hz, 3H); 13C NMR (67.5 MHz, CDCl3): d 171.9, 171.0,
170.5, 169.1, 133.6, 128.5, 70.5, 68.2, 61.9, 54.2, 52.2, 41.6, 40.7,
40.7, 39.5, 36.3, 33.5, 27.1, 16.7, 15.3, 11.5; IR (neat): 3340, 2967,
2931, 1713, 1662, 1504, 1216, 1164, 757 cm-1; [a]D21 = -57 (c 0.23,
MeOH), lit.6 [a]D = -59 (c 0.11, MeOH); HRMS (ESI-TOF): calcd
for [C21H34N3O6S2+H]+ 488.1889, found 488.1889.
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We thank EIWEISS Chemical Corporation for a generous supply
of EDCI·HCl
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