Total Syntheses and Biological Activities of Vinylamycin Analogues

Article abstract of DOI:10.1021/acs.jmedchem.6b01745

Natural depsipeptide vinylamycin was reported to be an antibiotic previously. Herein we report vinylamycin to be active against K562 leukemia cells (IC₅₀ = 4.86 μM) and be unstable in plasma (t_1/2 = 0.54 h). A total of 24 vinylamycin analogues with modification of the OH group and chiral centers were generated via a combinatorial approach. The lead compound 1a was subsequently characterized as having the following: no antimicrobial activity, significantly higher plasma stability (t_1/2 = 14.3 h), improved activity against K562 leukemia cells (IC₅₀ = 0.64 μM), and up to 75% cell inhibition without significant toxicities in K562 cells xenograft zebrafish model. Furthermore, compound 1a maintained its activity against the breast cancer cell line MCF-7 under hypoxic conditions. In comparison, the activity of gemcitabine in the same hypoxic in vitro model of MCF-7 cells was 15-fold lower. Therefore, the present results demonstrate that 1a has great potential as an anticancer agent.

Full text of DOI:10.1021/acs.jmedchem.6b01745

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Article
Total Syntheses and Biological Activities of Vinylamycin Analogs
Jinghan Wang, Beijia Kuang, Xiaoqian Guo, Jianwei Liu, Yahui Ding, Jiangnan Li, Shende Jiang, Ying Liu,
Fang Bai, Luyuan Li, Quan Zhang, Xiao-Yu Zhu, Bo Xia, Chun-Qi Li, Liang Wang, Guang Yang, and Yue Chen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01745 • Publication Date (Web): 11 Jan 2017
Downloaded from http://pubs.acs.org on January 11, 2017
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Total Syntheses and Biological Activities of Vinylamycin Analogs
Jinghan Wang,† Beijia Kuang,† Xiaoqian Guo,†,‡ Jianwei Liu,† Yahui Ding,† Jiangnan
Li,§ Shende Jiang,§ Ying Liu,† Fang Bai,† Luyuan Li,† Quan Zhang, † XiaoꢀYu Zhu,⊥
Bo Xia,⊥ ChunꢀQi Li,⊥ Liang Wang,† ,* Guang Yang,†,* and Yue Chen†,*
†The State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, and
Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin
300071, People's Republic of China
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‡Highꢀthroughput Molecular Drug Discovery Center, Tianjin International Joint
Academy of BioMedicine, Tianjin 300457, People's Republic of China
§School of Pharmaceutical Science and Technology, Tianjin University, Tianjin,
300072, People’s Republic of China
⊥Hunter Biotechnology, Inc, F1A, building 5, No. 88 Jiangling Road, Binjiang Zone,
Hangzhou, Zhejiang Province, 310051, People’s Republic of China
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Abstract
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Natural depsipeptide vinylamycin was reported to be an antibiotic previously. Herein,
we report vinylamycin to be active against K562 leukemia cells (IC50 = 4.86 ꢁM), and
be unstable in plasma (t1/2 = 0.54 h). A total of 24 vinylamycin analogs with
modification of the OH group and chiral centers were generated via a combinatorial
approach. The lead compound 1a was subsequently characterized as having: no
antiꢀmicrobial activity, significantly higher plasma stability (t1/2 = 14.3 h), improved
activity against K562 leukemia cells (IC50 = 0.64 ꢁM), and up to 75% cell inhibition
without significant toxicities in K562 cells xenograft zebrafish model. Furthermore,
compound 1a maintained its activity against the breast cancer cell line MCFꢀ7 under
hypoxic conditions. In comparison, the activity of paclitaxel in the same hypoxic in
vitro model of MCFꢀ7 cells was 92ꢀfold lower. Therefore, the present results
demonstrate that 1a has great potential as an anticancer agent.
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Introduction
Natural depsipeptides that contain 4ꢀaminoꢀ2,4ꢀpentadienoate moieties include
rakicidins,1 vinylamycin,2 microtermolides,3, 4 and BEꢀ43547A1 (Scheme 1).5, 6 In
particular, rakicidin A has exhibited unique activity against chronic myelogenous
leukemia (CML) stem cells7 and hypoxiaꢀselective cytotoxicity in solid tumors.8, 9
These observations led to the total synthesis and structural determination of rakicidin
A,10, 11 as well as an investigation of its structureꢀactivity relationship (SAR).12
Recently, Poulsen and coꢀworks reported total synthesis of entꢀBEꢀ43547A1 and
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revealed it significant hypoxiaꢀselectivity against PANCꢀ1 cancer cell line.6
Unfortunately, rakicidin A was found to be highly unstable at room temperature10, 11
and the preparation of rakicidin A or its analogs involved more than 35 steps with an
overall yield of 0.17%.12
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Vinylamycin is a depsipeptide that was originally isolated as a metabolite of
Streptomyces sp. and was subsequently identified as an antibiotic by Takeuchi and
coꢀworkers.2 The two amino acids of vinylamycin were identified as Dꢀvaline (2R)
and Lꢀalanine (5S),2 and only recently were the three consecutive chiral centers in the
polyketide fragment determined to be 14R, 15R, and 16R via total syntheses in our
group.13 The total synthesis of vinylamycin was accomplished with an overall yield of
3.7%,13 which is significantly higher than that for rakicidin A. However, similar to
rakicidin A, vinylamycin was also found to be unstable. Therefore,
4ꢀaminoꢀ2,4ꢀpentadienoate (APD) containing depsipepetides serve as good starting
point for drug development,14 thus, a series of vinylamycin analogs were synthesized
by using a combinatorial approach, and the activities of these analogs were
subsequently tested.
Results and Discussion
Synthesis of analogs
Scheme 2 shows the retroꢀsynthetic analysis that was used to obtain
TBDPSꢀprotected vinylamycins (compounds 1a-e). This synthetic sequence was
modified from our previously reported synthesis of vinylamycin.13 To obtain the
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precursor for the vinylamycin analogs (compound 2), several fragments were
synthesized individually: serinol derivative 3, Fmocꢀvaline 4 (with two isomers),
Fmocꢀalanine (with two isomers) or Fmocꢀglycine 5, and polyketide fragment 6 (with
three isomers).
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The polyketide fragments were obtained by a highly diastereoselective aldol
reaction between chiral aldehyde 9a15 and two camphorsultam derivatives, 8a and 8b
(Scheme 3). The corresponding aldol products, 10a and 10b, were hydrolyzed in an
aqueous THF solution in the presence of LiOH. The resulting carboxylic acids were
then converted into allyl esters 6a and 6b. Allyl ester 6c was synthesized as
previously reported13.
Direct esterification under previously optimized conditions between compounds
6a and 6c with Fmocꢀvaline 4a and 4b produced esters 12a, 12b, and 12c(Scheme
4).13 After deprotection of Fmoc in compounds 12a-c, the resulting amines were
coupled with compound 5a-d using EDCI and HOBt. Deprotection of the resulting
intermediates produced free carboxylic acids which were then subjected to coupling
reactions with serinol derivative 3 to generate the PMBꢀprotected precursor 13a-e.
After exchanging the PMB protecting group with TBDPS to obtain compounds 15a-e,
an additional two steps of deprotection were performed, and the intermediates were
cyclized using HATU and DIPEA to provide compounds 2a-e.13 The TBS protection
group in 2a-e was selectively deprotected under acidic conditions16, 17. Mesylation of
the resulting alcohol, followed by elimination with DBU, led to the production of
OꢀTBDPSꢀentꢀvinylamycin 1a, (14S, 15S, 16R)ꢀOꢀTBDPSꢀvinylamycin 1b,
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OꢀTBDPSꢀvinylamycin 1c, OꢀTBDPSꢀentꢀ5ꢀdemethylvinylamycin 1d, and OꢀTBDPS
entꢀ5ꢀdemethylꢀ6ꢀmethyl vinylamycin 1e.
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A series of Oꢀfunctionalized vinylamycin analogs with a 2S, 5R, 14S, 15S, 16R
configuration were synthesized using a combinatorial chemistry strategy. Compound
13a was used as the common intermediate for synthesis of a library of compounds
since it could be easily prepared in gramꢀscale (Scheme 5). Deprotection of the two
protection groups in 13a followed by macrolactamization, cyclic compound 16 was
delivered in good yield. However, selective PMB cleavage to obtain compound 17
proved to be problematic. In an aqueous DCM suspension in the presence of DDQ,
both the TBS and PMB groups were removed within 30 min18, 19, and an undesired
compound 18 was produced. Lowering of the reaction temperature to –10 ºC did not
improve the selectivity of the reaction. Therefore, alternative fragments 24a-b,
TBDPSꢀprotected serinol derivatives, were used instead of TBSꢀprotected derivatives.
Briefly, aldehydes 21a-b were prepared as previously described,20 while compounds
23a-23b were obtained through the Wittig reaction (Scheme 6). Compounds 23a-b
were then treated with TFA to afford serinol derivatives 24a-b.
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Removal of the allyl ester group of 25a-b were followed by coupling with
fragments 24a-b to obtain cyclization of precursors 26a-b. After further deprotection
of the allyl group and Fmoc group, the resulting compounds were subjected directly to
a macrolactamization step, and cyclic compounds 27a-b were synthesized in good
yield. This time, TBDPS was very stable under DDQ oxidation condition to provide
compound 29, and functionalization of the primary hydroxyl group was
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straightforward. Thus, esterification of 29 delivered analogs 31a-h. After all of the
analogs were exposed to TBAF to produce primary alcohols (32a-h), the resulting
hydroxyl groups were activated by MsCl and eliminated with DBU respectively,
vinylamycin analogs 33a-h were produced, and OꢀPMB vinylamycin analogs
28a-28b were also obtained with successive deprotection of TBDPS and elimination
(Scheme 7).
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As shown in Scheme 8, TBS, TIPS and TES silyl ether vinylamycin analogs were
synthesized. Briefly, compound 27a was treated with TBAF/HOAc to obtain
compound 34, and mesylation of alcohol 34 afforded compound 35. Deprotection of
PMB in compound 35 with DDQ, followed by reaction with 36aꢀ36c and imidazole,
and elimination by DBU successively, silyl ether analogs 37aꢀ37c were obtained.
Bn ether analogs were also synthesized (Scheme 9). Coupling of alcohol 6b and
acid 4a under DIC and DMAP condition provided ester 38. After deprotection of
Fmoc in compounds 38, the resulting amine was coupled with compound 5a using
EDCI and HOBt. Deprotection of the resulting intermediate produced free carboxylic
acid, which was subjected to coupling reaction with serinol derivative 24a to generate
the cyclization precursor 39. After further deprotection of the allyl group and Fmoc
group, the resulting compound was subjected directly to a macrolactamization step,
which provided us compound 40. Compound 40 was exposed to TBAF to produce
primary alcohols 41, and the OH group was activated by MsCl and eliminated with
DBU to produce analog 42. PMB ether of micortermolide A 44 was obtained through
activation of precursor 43 by MsCl and elimination with DBU.
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Activities of the synthesized compounds against the CML cell line, K562
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Twentyꢀfour analogs were evaluated for their effects on viability of the CML cell line
K562 (Table 1). In addition, imatinib was introduced as a positive control, and the
natural product, vinylamycin, was included for comparison. The natural product
vinylamycin exhibited moderate potency against the K562 cells (IC50 = 4.86 ꢁM).
Microtermoides A had no inhibitory activity on K562 cell line (IC50 > 50 ꢁM), while
all of the TBDPS analogs exhibited stronger activity. For example, 16ꢀepiꢀOꢀTBDPS
vinylamycin 1f (IC50 = 1.78 ꢁM) was 3 times more potent than vinylamycin, while
OꢀTBDPSꢀvinylamycin 1c and the Nꢀmethyl group removed analog 1d exhibited
4ꢀfold and 2ꢀfold greater potency compared with vinylamycin (IC50 = 1.27 ꢁM and
2.31 ꢁM, respectively). The polyketide fragment analog with reversed chirality 1b,
OꢀTBDPSꢀentꢀvinylamycin 1a, and OꢀTBDPS entꢀ5ꢀdemethylꢀ6ꢀmethylꢀvinylamycin
1e exhibited even higher potency against the K562 cell line (IC50 = 0.88 ꢁM, 0.64 ꢁM,
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and 0.4 ꢁM, respectively), while (14S, 15S, 16R)ꢀOꢀPMBꢀvinylamycin 28b (IC50
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4.6 ꢁM) demonstrated comparable activity with vinylamycin, and
OꢀPMBꢀentꢀvinylamycin 28a (IC50 = 2.49 ꢁM) was more potent than vinylamycin
and 28b. PMB analogs 28a and 28b were less potent than their corresponding TBDPS
analogs 1a and 1b.
Regarding the ester analogs 33a-h, most exhibited lower antiꢀleukemia activity
compared with the TBDPS ether analogs. Specifically, 10ꢀundecynoic acid (which
formed ester 33a) exhibited an IC50 of 4.41 ꢁM, which was similar to the IC50 of
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vinylamycin. The ester with a shorter side chain, compound 33b, exhibited about
twoꢀfold weaker inhibition activity (IC50 = 8.00 ꢁM) compared with 33a. Interestingly,
the antiꢀCML activity of the greasy fatty acid ester 33e was abolished (> 50 ꢁM).
Thus, various of organic acids were employed to generate ester analogs. The
antiꢀCML activity of compound 33c which contains a triꢀphenyl group (IC50 = 3.79
ꢁM) was comparable to that of vinylamycin. As to the Boc group protected
phenylalanine analog 33f, its antiꢀCML activity exhibited a moderate decrease to IC50
= 5.95 ꢁM. Compounds 33d and 33g, which included a diamantane group and a
phosphonate side chain, respectively, exhibited similar potency. When Boc group as
the protection group, i.e. compound 33h, its inhibitory activity against K562 was also
decreased to IC50 = 5.43 ꢁM. The inhibitory activities of other silyl ether analogs with
TBS, TIPS and TES were assayed. TBS ether analog (37a) and TES ether analog (37c)
show the moderate decreased potency (IC50 = 2.31 and 1.54 ꢁM, respectively) than
TBDPS ether analog. TIPS ether exhibited the similar potency (IC50 = 1.05 ꢁM) with
TBDPS ether. OꢀBnꢀentꢀvinylamycin 42 exhibited similar inhibitory activity (IC50 =
3.06 ꢁM) with OꢀPMBꢀentꢀvinylamycin 28a. OꢀPMBꢀMicrotermolides A 44 showed
weak inhibitory activity (IC50 = 13.69 ꢁM), while Microtermoides A was basically
inactive (IC50 > 50 ꢁM). The potency of the OH group precursors of vinylamycin 32a,
34 and 41 have much lower activities (17.88, 21.36 and 33.65 ꢁM respectively) than
vinylamycin.
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Antimicrobial activity of the synthesized compounds against Staphylococcus
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The minimal inhibitory concentration (MIC) of the various synthesized compounds
against Staphylococcus aureus were determined, with ciprofloxacin and vinylamycin
included as positive controls (Table 2). None of the tested synthetic vinylamycin
analogs exhibited antimicrobial activity (MIC > 64 ꢁg/mL).
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TBDPS analog 1a and ester analog 33a induce apoptosis in K562 cells
Compounds 1a and 33a were further analyzed in an apoptosis study by annexin
VꢀFITC/PI double staining. Based on these results, it is hypothesized that the ability to
induce apoptosis by 1a and 33a is the cause of the reduced proliferation induced by
the compounds as seen from Figure 1.
Chemical stability
In general, vinylamycin exhibited poor chemical stability. In addition, vinylamycin
was very sensitive to acids, bases, and temperature. However, a freezeꢀdried powder
of pure vinylamycin that was stored at –20 ºC under an argon atmosphere showed
minimal decomposition after 3 days. In contrast, all of the Oꢀfunctionalized
vinylamycin analogs were much more stable, and most could be stored at –20 ºC for
at least one month without apparent decomposition. These results indicate that the
introduction of an ester or silyl group for the free OH group significantly improves the
chemical stability of vinylamycin.
Plasma stability
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Plasma stability of vinylamycin and analogs 1a and 1e in the plasma of SD rat were
tested. Vinylamycin had the shortest halfꢀlife (0.54 h) in plasma among the three
compounds. In comparison, the plasma stability of analogs 1a and 1e were
significantly improved, with halfꢀlives of 14.3 h and 20.1 h, respectively. Thus,
structural modifications at the free hydroxyl group appear to improve the plasma
stability of vinylamycin.
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Cytotoxicity of vinylamycin analogs under two oxygenation conditions and index
of hypoxia selectivity
Hypoxia selectivity against the breast cancer cell line, MCFꢀ7, was evaluated for
compounds 1a, 1b, 1c, 1d, 1f, and 28b (Table 3). The inhibitory activities of
gemcitabine and paclitaxel against MCFꢀ7 cells under hypoxic conditions were
15.6ꢀfold and 92ꢀfold lower than that under normoxic conditions, respectively. In
contrast, the inhibitory activities of the analogs under hypoxic conditions were
essentially maintained, with selectivity index values ranging from 0.78–1.37. Taken
together, these results indicate that these analogs of vinylamycin can inhibit breast
cancer cells under hypoxic conditions. The inhibitory activities of rakicdin A,
vinylamycin, and methyl ester of rakicidin A have higher hypoxia selectivity
(selectivity index = 0.27, 0.74 and 0.38 respectively). Microtermolides A had no
inhibitory activity under hypoxic conditions and normoxic conditions.
Safety and inhibitory activity in xenograft zebrafish model.
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The safety and inhibitory activity of compound 1a against K562 cells in vivo were
assayed with xenografted zebrafish model (Table 4 and Figure 2)21. Compound 1a
was safe to zebrafish even at the saturated concentration of 50 ꢁg/mL, which
indicated that the toxicity of compound 1a was not higher than imatinib. The
inhibition of cancer cells of compound 1a were 66%, 70% and 75% respectively in
concentration of 5.6 ꢁg/mL, 16.7 ꢁg/mL and 50 ꢁg/mL. It suggests that compound 1a
have comparable inhibitory activity as imatinib (71% inhibition in 50 ꢁg/mL) in vivo.
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Conclusions
Initially, vinylamycin was reported to be an antibiotic natural product.2 Herein we
report its activity against the growth of CML cell line K562, and its low stability in rat
plasma. 24 vinylamycin analogs with modification of the OH group and chiral centers
were synthesized via a highly efficient combinatorial approach, which was modified
from our previously reported total synthesis of vinylamycin.11 Biological assays that
were performed for these analogs identified that the TBDPS ether analogs
(compounds 1a-f) were more potent than the PMB ether analog (compound 28) and
the ester analogs (33a-g) (Table 1). Among the TBDPS ether analogs,
OꢀTBDPSꢀentꢀvinylamycin (compound 1a) did not exhibit antiꢀmicrobial activity
(Table 2), yet was still able to induce apoptosis of K562 cells. Finally, compound 1a
maintained activity against the breast cancer cell MCFꢀ7 under hypoxic conditions,
while the activity of paclitaxel was found to decrease 92ꢀfold under the hypoxia
conditions (Table 3). These results are of particular interest given that tumor cells
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under hypoxic conditions are generally resistant to conventional chemo/radiotherapy,
and most compounds are less potent in hypoxic conditions22ꢀ24. These observations, in
addition to the increased stability of compound 1a at room temperature and in rat
plasma, and good in vivo efficacy in K562 cells xenografted zebrafish models, suggest
that compound 1a is a promising antiꢀcancer agent that warrants further investigation.
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EXPERIMENTAL SECTION
1. Chemistry. General. Unless otherwise mentioned, all reactions were carried out
under an argon atmosphere with dry solvents under anhydrous conditions. The used
solvents were purified and dried according to common procedures. Yields refer to
chromatographically and spectroscopically (1H NMR) homogeneous materials, unless
otherwise stated. Reagents were purchased at the highest commercial quality and used
without further purification, unless otherwise stated. Purity testing was done by means
of analytical HPLC on a Shimadzu LDꢀ20A system with an ODSꢀC18 column (4.6 ×
150 mm, 5 ꢁm) eluted at 1 mL/min with MilliꢀQ water and CH3CN. All tested
compounds were > 95% pure. FTIR spectra were obtained with a Bruker Tensor 27
instrument. All IR samples were reported in wave numbers (cmꢀ1). NMR spectra were
recorded with a 400 MHz or 600 MHz spectrometer using CDCl3, DMSOꢀd6 or
CD3OD. Data are reported as follows: chemical shift, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet), coupling constants and integration.
(3S,6R,14S,15S,E)-14-(2-(tert-Butyldiphenylsilyloxy)ethyl)-3-isopropyl-6-methyl-11-
methylene-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetr
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To a solution of compound 2a (0.10 g, 0.11 mmol) in MeOH (1.5 mL) was added a
solution of camphor sulfonic acid in MeOH (0.40 mL, 2 M, 0.80 mmol) at ꢀ 10 ºC.
The mixture was stirred at this temperature for 3 h, and then quenched with saturated
aqueous NaHCO3 (10 mL). The organic solvent was removed under reduced pressure
and the residue was extracted with ethyl acetate (3 × 20 mL). The combined organic
layers were dried over Na2SO4 and concentrated. The residue was purified by column
chromatography on silica gel chromatography (CH2Cl2: MeOH = 100: 3 to 100: 5) to
obtain a white solid.
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To a solution of obtained solid (33 mg, 0.044 mmol) in THF (2 mL), triethyl amine
(36 ꢁL, 0.260 mmol) and methane sulfonyl chloride (10 ꢁL, 0.13 mmol) were added
at 0 ºC. After stirred for 30 min, the reaction solution was quenched by addition of
water (0.1 mL), dried over Na2SO4, filtered and concentrated under reduced pressure.
The resulting crude product was dissolved in THF (2 mL). To the resulting solution
was added DBU (0.11 g, 0.70 mmol) at 20 ºC. After stirred for 2 h, the reaction was
quenched by addition of 1 % HCl (5 mL). The aqueous phase was extracted with ethyl
acetate (3 × 30 mL). The combined organic phases were washed with saturated
aqueous NaHCO3 (3 × 3 mL), dried over Na2SO4 and concentrated under reduced
pressure. The crude product was purified by column chromatography on silica gel
(CH2Cl2: MeOH = 30: 1) to obtain 1a (14 mg, 17% for three steps) as a white solid.
[α]20 = − 97.4 (c = 0.11, DMSO); νmax (KBr): 3293, 2930, 2861, 1736, 1673, 1522,
D
1
1464, 1522, 1256, 1106, 982, 893, 828 cmꢀ1; H NMR (400 MHz, DMSOꢀd6) δ 8.23
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(d, J = 9.8 Hz, 1H), 7.66 – 7.56 (m, 5H), 7.44 (dq, J = 14.0, 7.1 Hz, 6H), 6.85 (d, J =
15.2 Hz, 1H), 6.20 (d, J = 15.2 Hz, 1H), 5.34 (s, 1H), 5.24 – 5.18 (m, 2H), 4.32 – 4.24
(m, 1H), 4.22 – 4.16 (m, 1H), 3.73 – 3.58 (m, 2H), 2.92 – 2.82 (m, 1H), 1.99 – 1.87
(m, 1H), 1.85 – 1.63 (m, 3H), 1.27 (m, 13H), 0.99 (s, 9H), 0.91 (d, J = 6.5 Hz, 3H),
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0.84 (m, 9H). C NMR (100 MHz, DMSOꢀd6) δ 172.7, 171.0, 168.8, 166.1, 138.7,
137.5, 135.0, 133.1, 132.9, 129.8, 127.85, 118.9, 116.2, 75.9, 61.4, 57.5, 50.9, 44.7,
33.6, 33.4, 32.2, 31.9, 31.1, 28.8, 26.6, 22.0, 19.3, 18.6, 18.2, 18.1, 13.9, 13.2.
HRMS–MALDI (m/z): [M + Na]+ calcd for C42H61N3NaO6Si+, 754.4222, found
754.4218. HPLC purity: 97.2%.
(3R,6S,14S,15S,E)-14-(2-(tert-Butyldiphenylsilyloxy)ethyl)-3-isopropyl-6-methyl-11-
methylene-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetr
aone (1b)
The titled compound 1b was obtained following the general procedure described for
1a. Flash column chromatography eluent (CH2Cl2: MeOH = 30: 1); yield, 19% for
three steps; white powder; [α]20 = − 24.1 (c = 0.1, DMSO); νmax (KBr): 3273, 3051,
D
2927, 2860, 1735, 1671, 1622, 1514, 1464, 1370, 1198, 1105, 990, 821 cmꢀ1; 1H NMR
(400 MHz, DMSOꢀd6) δ 8.61 (d, J = 9.1 Hz, 1H), 8.23 (s, 1H), 7.70 (d, J = 5.2 Hz,
1H), 7.62 (d, J = 7.6 Hz, 4H), 7.51 – 7.37 (m, 6H), 6.72 (d, J = 15.7 Hz, 1H), 6.09 (d,
J = 15.8 Hz, 1H), 5.60 (s, 1H), 5.34 (s, 1H), 5.10 – 5.04 (m, 1H), 4.54 (dd, J = 9.8, 5.6
Hz, 1H), 4.10 – 4.02 (m, 1H), 3.70 – 3.59 (m, 2H), 3.11 – 3.00 (m, 1H), 1.97 (d, J =
6.3 Hz, 1H), 1.76 – 1.67 (m, 1H), 1.65 – 1.56 (m, 1H), 1.42 – 1.32 (m, 2H), 1.29 –
13
1.12 (m, 14H), 0.99 (s, 8H), 0.87 – 0.79 (m, 9H), 0.74 (d, J = 6.6 Hz, 3H). C NMR
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(100 MHz, DMSOꢀd6) δ 173.4, 170.9, 170.4, 167.7, 137.3, 136.9, 135.0, 135.0, 132.9,
132.9, 129.9, 127.9, 127.8, 120.6, 113.6, 76.8, 60.8, 55.9, 51.2, 45.9, 34.7, 32.4, 31.5,
31.0, 30.3, 28.9, 26.6, 25.6, 22.0, 19.1, 18.7, 18.5, 17.3, 14.5, 13.9. HRMS–MALDI
(m/z): [M + Na]+ calcd for C42H61N3NaO6Si+, 754.4222, found 754.4219. HPLC
purity: 97.9%.
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(3R,6S,14R,15R,E)-14-(2-(tert-Butyldiphenylsilyloxy)ethyl)-3-isopropyl-6-methyl-11
-methylene-15-((S)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetr
aone (1c)
The titled compound 1c was obtained following the general procedure described for
19a. Flash column chromatography eluent (CH2Cl2: MeOH = 30: 1); yield, 25% for
three steps; white powder; [α]20D = + 88.3 (c = 0.12, DMSO); νmax (KBr): 3292, 2929,
1
2860, 1737, 1673, 1524, 1465, 1258, 1106, 983, 823 cmꢀ1; H NMR (400 MHz,
DMSOꢀd6) δ 8.23 (d, J = 9.8 Hz, 1H), 7.66 – 7.56 (m, 5H), 7.44 (dq, J = 14.0, 7.1 Hz,
6H), 6.85 (d, J = 15.2 Hz, 1H), 6.20 (d, J = 15.2 Hz, 1H), 5.34 (s, 1H), 5.24 – 5.18 (m,
2H), 4.32 – 4.24 (m, 1H), 4.22 – 4.16 (m, 1H), 3.73 – 3.58 (m, 2H), 2.92 – 2.82 (m,
1H), 1.99 – 1.87 (m, 1H), 1.85 – 1.63 (m, 3H), 1.27 (m, 13H), 0.99 (s, 9H), 0.91 (d, J
13
= 6.5 Hz, 3H), 0.84 (m, 9H). C NMR (100 MHz, DMSOꢀd6) δ 172.7, 171.0, 168.8,
166.1, 138.7, 137.5, 135.0, 133.1, 132.9, 129.8, 127.85, 118.9, 116.2, 75.9, 61.4, 57.5,
50.9, 44.7, 33.6, 33.4, 32.2, 31.9, 31.1, 28.8, 26.6, 22.0, 19.3, 18.6, 18.2, 18.1, 13.9,
13.2. HRMS–MALDI (m/z): [M + Na]+ calcd for C42H61N3NaO6Si+, 754.4222, found
754.4216. HPLC purity: 96.7%
(3S,14S,15S,E)-14-(2-(tert-Butyldiphenylsilyloxy)ethyl)-3-isopropyl-11-methylene-1
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5-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (1d)
The titled compound 1d was obtained following the general procedure described for
1a. Flash column chromatography eluent (CH2Cl2: MeOH = 30: 1); yield, 20% for
three steps; white powder; [α]20D = − 52.3 (c = 0.13, DMSO); νmax (KBr): 3315, 2927,
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2860, 1721, 1680, 1526, 1463, 1364, 1264, 1105, 983, 859, 826 cmꢀ1; H NMR (400
MHz, DMSOꢀd6) δ 8.86 (s, 1H), 8.22 (d, J = 9.7 Hz, 1H), 7.76 – 7.56 (m, 5H), 7.51 –
7.33 (m, 6H), 6.83 (d, J = 15.2 Hz, 1H), 6.17 (d, J = 15.2 Hz, 1H), 5.34 (s, 1H), 5.23 –
5.15 (m, 2H), 4.27 – 4.20 (m, 1H), 4.11 (dd, J = 17.4, 4.9 Hz, 1H), 3.73 – 3.49 (m,
3H), 3.01 – 2.82 (m, 1H), 1.92 (dd, J = 13.5, 6.6 Hz, 1H), 1.82 – 1.63 (m, 3H), 1.38 –
1.15 (m, 12H), 0.99 (s, 9H), 0.93 – 0.79 (m, 14H). 13C NMR (100 MHz, DMSOꢀd6) δ
171.0, 168.9, 168.8, 166.9, 138.3, 137.5, 135.0, 133.1, 132.9, 131.6, 129.8, 128.6,
127.9, 118.8, 116.1, 75.9, 67.4, 61.4, 57.5, 44.6, 44.2, 38.1, 33.6, 33.4, 32.3, 31.6,
31.1, 29.8, 28.8, 28.3, 26.6, 23.2, 22.4, 22.0, 19.3, 18.6, 18.3, 13.9, 13.2, 10.8.
HRMS–MALDI (m/z): [M + Na]+ calcd for C41H59N3NaO6Si+, 740.4065, found
740.4069. HPLC purity: 98.2%.
(3S,14S,15S,E)-14-(2-(tert-Butyldiphenylsilyloxy)ethyl)-3-isopropyl-7-methyl-11-me
thylene-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraon
e (1e)
The titled compound 1e was obtained following the general procedure described for
1a. Flash column chromatography eluent (CH2Cl2: MeOH = 30: 1); yield, 18% for
three steps; white powder; [α]20D = − 156.7 (c = 0.09, DMSO); νmax (KBr): 2959, 2860,
1731, 1689, 1611, 1528, 1470, 1259, 1107, 982, 820 cmꢀ1; H NMR (400 MHz,
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DMSOꢀd6) δ 8.93 (s, 1H), 8.27 (d, J = 9.6 Hz, 1H), 7.70 – 7.56 (m, 4H), 7.52 – 7.37
(m, 6H), 6.81 (d, J = 14.8 Hz, 1H), 6.40 (d, J = 14.8 Hz, 1H), 5.33 (s, 1H), 5.24 – 5.12
(m, 2H), 4.61 (d, J = 17.4 Hz, 1H), 4.20 (dd, J = 9.5, 7.7 Hz, 1H), 3.85 (d, J = 17.4 Hz,
1H), 3.73 – 3.55 (m, 2H), 3.36 (s, 2H), 3.00 (s, 3H), 1.99 – 1.83 (m, 1H), 1.81 – 1.73
(m, 2H), 1.70 – 1.64 (m, 1H), 1.52 (s, 1H), 1.35 – 1.10 (m, 16H), 0.99 (s, 9H), 0.94 –
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0.83 (m, 12H). C NMR (100 MHz, DMSOꢀd6) δ 171.2, 168.7, 168.4, 165.7, 138.4,
137.7, 135.0, 133.1, 132.9, 129.8, 127.9, 118.8, 116.0, 76.1, 61.5, 57.7, 52.3, 45.4,
44.5, 36.6, 33.6, 33.4, 32.3, 31.7, 31.1, 28.8, 26.6, 22.0, 19.3, 18.7, 18.4, 13.9, 13.2,
8.7. HRMS–MALDI (m/z): [M + Na]+ calcd for C42H61N3NaO6Si+, 754.4222, found
754.4214. HPLC purity: 95.7%.
(3S,6R,11R,14S,15S,E)-11-((tert-Butyldimethylsilyloxy)methyl)-14-(2-(tert-butyldip
henylsilyloxy)ethyl)-3-isopropyl-6-methyl-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyc
lopentadec-9-ene-2,5,8,13-tetraone (2a)
The compound 15a (0.370 g, 0.330 mmol) and Pd(PPh3)4 (76 mg, 0.0660 mmol) was
dissolved in anhydrous THF (3.0 mL), and Nꢀmethyl aniline (72 ꢁL, 0.660 mmol) was
added. After stirred at room temperature for 1.5 h, the reaction mixture was
concentrated under reduced pressure. The residue was purified by column
chromatography on silica gel (petroleum ether: ethyl acetate = 20: 1 to 1: 1) to afford
the acid as pale yellow foam. The obtained acid was dissolved in CH2Cl2 (2 mL) and
diethyl amine (1 mL), the reaction mixture was stirred at room temperature for 3 h,
and then the solvent was removed under reduced pressure to afford the crude amino
acid. The mixture was dissolved in THF (270 mL), and then DIPEA (0.75 mL, 4.30
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mmol) and HATU (0.820 g, 2.20 mmol) was added successively at 0 ºC. After stirred
at room temperature for 12 h, the solvent was removed under reduced pressure, and
then the residue was dissolved in ethyl acetate (200 mL) and washed successively
with 1% HCl, saturated aqueous NaHCO3, brine, dried over Na2SO4. The solution was
filtrated and concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel (CH2Cl2/MeOH = 100: 1 to 100: 4) to obtain the
cyclic peptide 2a (0.171 g, 60 % for three steps) as a white powder. [α]20D = − 36.7 (c
= 0.15, DMSO); νmax (KBr): 3403, 2931, 2860, 1732, 1677, 1531, 1464, 1260, 1109,
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986, 845 cmꢀ1; H NMR (400 MHz, CD3OD) δ 7.70 – 7.61 (m, 5H), 7.46 – 7.37 (m,
6H), 6.95 (d, J = 15.1 Hz, 1H), 6.05 (d, J = 15.1 Hz, 1H), 5.45 (d, J = 10.6 Hz, 1H),
4.65 (s, 1H), 4.42 – 4.32 (m, 2H), 3.83 – 3.68 (m, 2H), 3.56 – 3.46 (m, 2H), 2.89 –
2.72 (m, 1H), 2.11 – 1.96 (m, 1H), 1.81 (d, J = 6.5 Hz, 2H), 1.62 – 1.51 (m, 1H), 1.47
– 1.39 (m, 4H), 1.39 – 1.16 (m, 15H), 1.04 (s, 9H), 0.97 – 0.78 (m, 28H), 0.02 (s, 6H).
13C NMR (100 MHz, CD3OD) δ 175.1, 174.7, 170.6, 169.1, 145.8, 136.8, 136.7,
134.8, 134.7, 131.1, 131.1, 129.0, 119.7, 77.5, 65.9, 62.7, 59.5, 53.7, 52.7, 46.5, 35.4,
35.2, 33.9, 33.6, 32.95, 30.67, 28.42, 27.5, 26.5, 23.7, 20.3, 20.0, 19.2, 19.0, 14.6,
+
13.8, ꢀ5.1, ꢀ5.1. HRMS–MALDI (m/z): [M + Na]+ calcd for C48H77N3NaO7Si2 ,
886.5192, found 886.5196.
(3R,6S,11S,14S,15S,E)-11-((tert-Butyldimethylsilyloxy)methyl)-14-(2-(tert-butyldiph
enylsilyloxy)ethyl)-3-isopropyl-6-methyl-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacycl
opentadec-9-ene-2,5,8,13-tetraone (2b)
The titled compound 2b was obtained following the general procedure described for
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2a. Flash column chromatography eluent (CH2Cl2: MeOH = 100: 1 to 100: 4); yield,
49% for three steps; white powder; [α]20D = − 35.1 (c = 0.1, DMSO); νmax(KBr): 3286,
3064, 2927, 2859, 1735, 1665, 1521, 1463, 1435, 1365, 1268, 1233, 1105, 978, 821
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cmꢀ1; H NMR (400 MHz, CDCl3) δ 7.66 – 7.58 (m, 4H), 7.45 – 7.33 (m, 6H), 6.48
(dd, J = 15.0, 9.7 Hz, 1H), 6.10 (d, J = 15.0 Hz, 1H), 5.05 (d, J = 8.1 Hz, 1H), 4.78 (d,
J = 6.4 Hz, 1H), 4.59 (s, 1H), 4.41 (s, 1H), 4.28 (s, 1H), 3.80 – 3.58 (m, 4H), 2.38 –
1.96 (m, 2H), 1.38 – 1.19 (m, 14H), 1.03 (s, 9H), 0.94 (d, J = 6.6 Hz, 3H), 0.84 (d, J =
13
15.3 Hz, 20H), 0.06 (s, 6H). C NMR (100 MHz, acetoneꢀd6) δ 174.2, 172.2, 171.5,
171.3, 168.3, 142.2, 136.4, 136.3, 134.4, 134.2, 130.7, 128.7, 128.7, 123.0, 78.9, 65.7,
61.8, 57.3, 52.7, 46.3, 36.3, 33.8, 32.6, 32.5, 31.2, 27.3, 27.0, 26.3, 23.4, 19.8, 19.4,
18.8, 17.7, 15.5, 14.4, ꢀ5.1. HRMS–MALDI (m/z): [M + Na]+ calcd for
+
C48H77N3NaO7Si2 , 886.5192, found 886.5197.
(3R,6S,11S,14R,15R,E)-11-((tert-Butyldimethylsilyloxy)methyl)-14-(2-(tert-butyldip
henylsilyloxy)ethyl)-3-isopropyl-6-methyl-15-((S)-octan-2-yl)-1-oxa-4,7,12-triazacyc
lopentadec-9-ene-2,5,8,13-tetraone (2c)
The titled compound 2c was obtained following the general procedure described for
2a. Flash column chromatography eluent (CH2Cl2: MeOH = 100: 1 to 100: 4); yield,
52% for three steps; white powder; [α]20D = + 31.3 (c = 0.2, DMSO); νmax (KBr): 3402,
2931, 2860, 1731, 1677, 1530, 1464, 1260, 1108, 986, 845 cmꢀ1; 1H NMR (400 MHz,
CD3OD) δ 7.70 – 7.61 (m, 5H), 7.46 – 7.37 (m, 6H), 6.95 (d, J = 15.1 Hz, 1H), 6.05
(d, J = 15.1 Hz, 1H), 5.45 (d, J = 10.6 Hz, 1H), 4.65 (s, 1H), 4.42 – 4.32 (m, 2H), 3.83
– 3.68 (m, 2H), 3.56 – 3.46 (m, 2H), 2.89 – 2.72 (m, 1H), 2.11 – 1.96 (m, 1H), 1.81 (d,
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J = 6.5 Hz, 2H), 1.62 – 1.51 (m, 1H), 1.47 – 1.39 (m, 4H), 1.39 – 1.16 (m, 15H), 1.04
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(s, 9H), 0.97 – 0.78 (m, 28H), 0.02 (s, 6H). C NMR (100 MHz, CD3OD) δ 175.1,
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174.7, 170.6, 169.1, 145.8, 136.8, 136.7, 134.8, 134.7, 131.1, 131.1, 129.0, 119.7,
77.5, 65.9, 62.7, 59.5, 53.7, 52.7, 46.5, 35.4, 35.2, 33.9, 33.6, 32.95, 30.67, 28.42,
27.5, 26.5, 23.7, 20.3, 20.0, 19.2, 19.0, 14.6, 13.8, ꢀ5.1, ꢀ5.1. HRMS–MALDI (m/z):
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[M + Na]+ calcd for C48H77N3NaO7Si2 , 886.5192, found 886.5196.
(3S,11R,14S,15S,E)-11-((tert-Butyldimethylsilyloxy)methyl)-14-(2-(tert-butyldiphen
ylsilyloxy)ethyl)-3-isopropyl-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9
-ene-2,5,8,13-tetraone (2d)
The titled compound 2d was obtained following the general procedure described for
2a. Flash column chromatography eluent (CH2Cl2: MeOH = 100: 1 to 100: 4); yield,
58% for three steps; white powder; [α]20D = − 56.4 (c = 0.09, DMSO); νmax (KBr):
1
3320, 2929, 2864, 1733, 1673, 1452, 1256, 1054, 976, 914, 850 cmꢀ1; H NMR (400
MHz, CD3OD) δ 7.73 – 7.63 (m, 4H), 7.49 – 7.37 (m, 6H), 6.91 (dd, J = 15.1, 2.6 Hz,
1H), 6.00 (d, J = 15.1 Hz, 1H), 5.45 (d, J = 10.7 Hz, 1H), 4.66 (s, 1H), 4.45 (d, J = 6.7
Hz, 1H), 4.10 (d, J = 17.4 Hz, 1H), 3.86 – 3.72 (m, 3H), 3.66 – 3.45 (m, 2H), 2.78 (dd,
J = 13.6, 9.1 Hz, 1H), 2.06 (dd, J = 13.4, 6.7 Hz, 1H), 1.82 (d, J = 7.0 Hz, 2H), 1.67 –
1.54 (m, 1H), 1.45 – 1.18 (m, 13H), 1.04 (s, 9H), 1.00 – 0.82 (m, 22H), 0.03 (s, 6H).
13C NMR (100 MHz, CD3OD) δ 174.5, 171.0, 170.9, 170.2, 145.1, 136.8, 136.7,
134.8, 134.7, 133.9, 133.2, 133.1, 131.1, 131.1, 130.1, 130.0, 129.0, 119.8, 77.3, 66.0,
62.7, 59.7, 59.6, 53.7, 46.5, 45.5, 39.0, 35.4, 35.2, 34.1, 33.2, 33.0, 30.7, 28.5, 27.6,
26.6, 23.8, 20.2, 20.0, 19.3, 19.0, 14.6, 14.0, ꢀ5.0, ꢀ5.0. HRMS–MALDI (m/z): [M +
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Na]+ calcd for C47H75N3NaO7Si2 , 872.5036, found 872.5041.
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(3S,11R,14S,15S,E)-11-((tert-Butyldimethylsilyloxy)methyl)-14-(2-(tert-butyldiphen
ylsilyloxy)ethyl)-3-isopropyl-7-methyl-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclop
entadec-9-ene-2,5,8,13-tetraone (2e)
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The titled compound 2e was obtained following the general procedure described for
2a. Flash column chromatography eluent (CH2Cl2: MeOH = 100: 1 to 100: 4); yield,
61% for three steps; white powder; [α]20 = − 43.1 (c = 0.13, DMSO); νmax (KBr):
D
3351, 2933, 2860, 1705, 1664, 1545, 1467, 1427, 1391, 1276, 1220, 1110, 1082, 912,
839 cmꢀ1; 1H NMR (400 MHz, DMSOꢀd6) δ 8.17 (d, J = 8.9 Hz, 1H), 7.77 (d, J = 8.2
Hz, 1H), 7.61 (d, J = 6.2 Hz, 3H), 7.52 – 7.33 (m, 6H), 6.69 (d, J = 14.9 Hz, 1H), 6.01
(d, J = 14.6 Hz, 1H), 5.22 (d, J = 10.4 Hz, 1H), 4.48 (s, 1H), 4.37 (d, J = 17.9 Hz, 1H),
4.19 (s, 1H), 3.85 (d, J = 18.0 Hz, 1H), 3.65 (d, J = 7.5 Hz, 2H), 3.41 (m, 2H), 2.96 (s,
3H), 2.70 (s, 1H), 1.89 (d, J = 6.2 Hz, 1H), 1.69 (s, 2H), 1.48 (s, 1H), 1.33 – 1.06 (m,
10H), 0.98 (s, 9H), 0.91 – 0.63 (m, 21H), – 0.02 (s, 6H). 13C NMR (100 MHz,
DMSOꢀd6) δ 171.1, 168.8, 168.0, 165.4, 142.2, 135.0, 135.0, 133.1, 129.1, 127.8,
119.0, 75.0, 64.4, 61.2, 57.3, 52.1, 51.2, 44.1, 36.5, 33.5, 32.3, 31.5, 31.1, 28.9, 26.6,
25.7, 22.0, 19.3, 18.6, 18.4, 17.9, 13.9, 13.2, ꢀ5.6. HRMS–MALDI (m/z): [M + Na]+
+
calcd for C48H77N3NaO7Si2 , 886.5192, found 886.5199.
(2S,3S,4R)-Allyl-3-hydroxy-2-(2-(4-methoxybenzyloxy)ethyl)-4-methyldecanoate
(6a)
To a solution of 10a (4.00 g 7.10 mmol) in THF/H2O (40 mL/13 mL) was added
LiOH•H2O (447 mg, 10.6 mmol) at room temperature. After being stirred for 4 h at
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this temperature, the reaction mixture acidified to pH = 4.0 with aqueous 10%
NaHSO4 and extracted with ethyl acetate (2 × 100 mL). The solvent was evaporated,
and the resulting mixture was directly used for the next step without further
purification.
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To a solution of crude acid 11a in DMF (12.0 mL) was added K2CO3 (750 mg, 5.41
mmol) and allyl bromide (600 mg, 4.92 mmol) at room temperature. The mixture was
stirred at room temperature for 10 h, and then H2O (100 mL) was added, and the
resultant mixture was extracted with diethyl ether (3 × 50 mL). The combined organic
extracts were dried over MgSO4, filtered and concentrated under reduced pressure.
The residue was purified with column chromatography on silica gel (petroleum ether:
ethyl acetate = 20:1) to afford compound 6a (1.6 g, 56% for two steps) as a colorless
oil. [α]20D = − 35.7 (c = 1.0, CHCl3). νmax(KBr): 3527, 2955, 2928, 2856, 1731, 1612,
1513, 1460, 1248, 1173, 1097, 1036, 987, 931, 820 cmꢀ1; 1H NMR (400 MHz, CDCl3)
δ 7.26 (d, J = 8.2 Hz, 2H), 6.89 (d, J = 8.2 Hz, 2H), 5.98 – 5.83 (m, 1H), 5.34 (d, J =
16.8 Hz, 1H), 5.25 (d, J = 10.4 Hz, 1H), 4.59 (qd, J = 13.1, 5.8 Hz, 2H), 4.43 (s, 2H),
3.82 (s, 3H), 3.61 – 3.39 (m, 3H), 2.89 – 2.77 (m, 1H), 2.45 (d, J = 8.2 Hz, 1H), 2.10
– 1.97 (m, 1H), 1.96 – 1.84 (m, 1H), 1.58 – 1.49 (m, 1H), 1.47 – 1.38 (m, 1H), 1.38 –
1.12 (m, 11H), 0.96 – 0.85 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 175.5, 159.2,
132.0, 130.4, 129.3, 118.6, 113.7, 77.4, 77.1, 76.7, 75.6, 72.7, 67.5, 65.3, 55.3, 45.6,
36.4, 33.6, 31.9, 30.0, 29.5, 27.0, 22.7, 14.1, 13.9.HRMS–ESI(m/z): [M + Na]+ calcd
+
for C24H38NaO5 , 429.2611, found 429.2619.
(2S,3S,4R)-Allyl-2-(2-(benzyloxy)ethyl)-3-hydroxy-4-methyldecanoate (6b)
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The titled compound 6b was obtained following the procedure described for 6a. Flash
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column chromatography (petroleum ether: ethyl acetate = 20: 1 to 15: 1); yield: 81%;
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colorless oil; [α]20 = − 38.2 (c = 1.3, CHCl3). νmax(KBr): 3523, 2955, 2926, 2857,
D
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1726, 1457, 1369, 1171, 1102, 987, 931 cmꢀ1; H NMR (400 MHz, CDCl3) δ 7.38 –
7.27 (m, 5H), 5.96 – 5.81 (m, 1H), 5.31 (d, J = 17.2 Hz, 1H), 5.22 (d, J = 10.4 Hz,
1H), 4.64 – 4.51 (m, 2H), 4.48 (s, 2H), 3.60 – 3.41 (m, 3H), 2.89 – 2.80 (m, 1H), 2.42
(d, J = 8.2 Hz, 1H), 2.10 – 1.98 (m, 1H), 1.95 – 1.81 (m, 1H), 1.53 (dt, J = 12.2, 6.3
13
Hz, 1H), 1.46 – 1.37 (m, 1H), 1.36 – 1.12 (m, 10H), 0.93 – 0.83 (m, 6H). C NMR
(100 MHz, CDCl3) δ 175.6, 138.4, 132.1, 128.5, 127.8, 127.7, 118.8, 75.8, 73.2, 68.0,
65.4, 45.8, 36.6, 33.7, 32.0, 30.2, 29.6, 27.1, 22.8, 14.2, 14.1. HRMS–MALDI (m/z):
[M + Na]+ calcd for C23H36NaO4 , 399.2506, found 399.2510.
+
(2S,3S,4R)-1-((6R,7R)-8,8-Dimethyl-2,2-dioxidohexahydro-1H-3,6-methanobenzo[c
]isothiazol-1-yl)-3-hydroxy-2-(2-((4-methoxybenzyl)oxy)ethyl)-4-methyldecan-1-one
(10a)
To a solution of compound 7a (4.00 g, 9.50 mmol) in CH2Cl2 (16 mL) was added
triethylamine (2.02 mL, 14.2 mmol) and TBSOTf (3.05 mL, 13.3 mmol). The reaction
mixture was stirred at room temperature for 18 h. The resulting solution was directly
used for the next step.
To a solution of aldehyde 9a (14.0 mL, 14.0 mmol) in CH2Cl2 (40 mL) was added
TiCl4 (1 M in CH2Cl2, 17.4 mL, 17.4 mmol) dropwise at ꢀ 78 ºC. After 5 min, the
solution of 8a above was added to the reaction mixture. The reaction was stirred at ꢀ
78 ºC for 3 h before saturated aqueous NaHCO3 (200 mL) was added. The aqueous
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phase was extracted with CH2Cl2 (2 × 100 mL). The combined organic phases were
dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel (petroleum ether: ethyl acetate =
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20:1 to 8:1) to obtain 10a (4.2 g, 78%) as a colorless oil. [α]20 = − 35.7 (c = 2.3,
D
CHCl3). νmax (KBr): 3527, 2955, 2928, 2856, 1731, 1612, 1513, 1460, 1248, 1173,
1
1097, 1036, 987, 931, 820 cmꢀ1; H NMR (400 MHz, CDCl3) δ 7.27 (d, J = 8.0 Hz,
1H), 6.85 (d, J = 8.0 Hz, 1H), 4.43 (d, J = 11.0 Hz, 1H), 4.34 (d, J = 11.0 Hz, 1H),
3.79 (s, 3H), 3.71 (s, 1H), 3.63 (t, J = 9.3 Hz, 1H), 3.55 – 3.46 (m, 3H), 3.39 (d, J =
13.8 Hz, 1H), 3.32 (s, 1H), 2.27 (d, J = 11.0 Hz, 2H), 2.15 (dt, J = 15.6, 7.9 Hz, 1H),
2.02 (dd, J = 13.8, 7.9 Hz, 1H), 1.92 – 1.79 (m, 3H), 1.78 – 1.67 (m, 2H), 1.62 (s, 1H),
1.46 – 1.36 (m, 1H), 1.34 – 1.19 (m, 12H), 1.16 (s, 3H), 0.94 (s, 3H), 0.86 (t, J = 6.3
Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 174.3, 159.2, 131.0, 129.6, 113.8, 72.6, 68.3,
65.8, 55.4, 53.4, 48.3, 47.8, 47.0, 44.8, 38.6, 35.5, 34.2, 33.0, 32.1, 29.7, 29.2, 27.4,
26.5, 22.8, 20.9, 20.1, 14.3, 12.5. HRMS–ESI(m/z): [M + Na]+ calcd for
C31H49NNaO6S+, 586.3173, found 586.3169.
(2S,3S,4R)-2-(2-(Benzyloxy)ethyl)-1-((6R,7R)-8,8-dimethyl-2,2-dioxidohexahydro-1
H-3a,6-methanobenzo[c]isothiazol-1-yl)-3-hydroxy-4-methyldecan-1-one (10b)
The titled compound 10b was obtained following the procedure described for 10a.
Flash column chromatography (petroleum ether: ethyl acetate = 20: 1 to 10: 1); yield:
67%; colorless oil; [α]20 = − 46.1 (c = 1.0, CHCl3). νmax(KBr): 3463, 2923, 2858,
D
1671, 1460, 1328, 1115, 801 cmꢀ1; 1H NMR (400 MHz, CDCl3) δ 7.36 – 7.09 (m, 5H),
4.44 (d, J = 11.5 Hz, 1H), 4.36 (d, J = 11.5 Hz, 1H), 3.65 (s, 1H), 3.62 – 3.53 (m, 1H),
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3.48 (t, J = 6.5 Hz, 2H), 3.43 (d, J = 13.8 Hz, 1H), 3.35 – 3.17 (m, 2H), 2.28 – 2.17
(m, 2H), 2.15 – 2.03 (m, 1H), 1.96 (dd, J = 13.8, 7.9 Hz, 1H), 1.86 – 1.74 (m, 3H),
1.72 – 1.63 (m, 1H), 1.61 – 1.51 (m, 1H), 1.37 (s, 1H), 1.28 – 1.14 (m, 11H), 1.10 (s,
3H), 0.88 (s, 3H), 0.81 (t, J = 7.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 174.2,
138.8, 128.3, 127.9, 127.5, 72.9, 68.5, 65.7, 53.3, 48.2, 47.8, 47.0, 44.7, 38.5, 35.5,
34.1, 33.0, 32.0, 29.6, 29.1, 27.4, 26.5, 22.8, 20.8, 20.0, 14.2, 12.4. HRMS–MALDI
(m/z): [M + Na]+ calcd for C30H47NNaO5S+, 556.3067, found, 556.3070.
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(2S,3S,4R)-Allyl-3-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-methylbut
anoyloxy)-2-(2-(4-methoxybenzyloxy)ethyl)-4-methyldecanoate (12a)
To a solution of acid 4a (2.99 g, 8.80 mmol) and 6a (1.79 g, 4.40 mmol) in CH2Cl2
(15 mL) was added DMAP (215 mg, 1.76 mmol) and DIC (2.70 mL, 17.6 mmol)
under argon atmosphere at 20 ºC. The reaction mixture was stirred for 18 h, and
diluted with CH2Cl2 (50 mL) then quenched with H2O (100 mL). The aqueous phase
was extracted with CH2Cl2 (3 × 50 mL). And the combined organic phases were dried
over Na2SO4 and concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel (petroleum ether/ethyl acetate = 20:1) to obtain
compound 12a (3.10 g, 96%) as colorless oil. [α]20 = − 82.9 (c = 1.0, CHCl3). IR
D
(KBr) νmax: 3443, 3377, 2952, 2931, 2860, 1740, 1734, 1612, 1512, 1463, 1366, 1248,
1173, 1092, 1038, 986, 931, 820 cmꢀ1; 1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 7.5
Hz, 2H), 7.58 – 7.51 (m, 2H), 7.32 (t, J = 7.3 Hz, 2H), 7.23 (td, J = 7.3, 2.8 Hz, 2H),
7.15 (d, J = 8.5 Hz, 2H), 6.78 (d, J = 8.5 Hz, 2H), 5.87 – 5.70 (m, 1H), 5.30 (d, J =
9.3 Hz, 1H), 5.22 (d, J = 16.9 Hz, 1H), 5.14 (d, J = 10.4 Hz, 1H), 5.11 – 5.07 (m, 1H),
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4.44 (d, J = 5.6 Hz, 1H), 4.30 (d, J = 5.5 Hz, 1H), 4.23 (dd, J = 9.3, 4.4 Hz, 1H), 4.16
(t, J = 7.1 Hz, 1H), 3.71 (s, 3H), 3.44 – 3.36 (m, 1H), 3.32 (m, 1H), 2.98 – 2.90 (m,
1H), 2.15 – 2.05 (m, 1H), 1.93 – 1.81 (m, 1H), 1.70 (s, 2H), 1.36 – 1.26 (m, 1H), 1.25
– 1.10 (m, 9H), 1.06 (d, J = 6.5 Hz, 3H), 0.91 (d, J = 6.8 Hz, 3H), 0.85 – 0.75 (m,
11H).13C NMR (100 MHz, CDCl3) δ 172.5, 171.3, 159.2, 156.3, 144.0, 143.8, 141.3,
132.0, 130.3, 129.3, 127.7, 127.1, 125.2, 120.0, 118.6, 113.8, 78.0, 72.7, 67.1, 65.4,
59.3, 55.3, 53.5, 47.2, 44.8, 42.2, 34.8, 33.4, 31.8, 31.0, 29.4, 26.9, 23.5, 22.6, 19.5,
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+
17.2, 14.1. HRMS–MALDI (m/z): [M + Na]+ calcd for C44H57NNaO8 , 750.3976,
found 750.3973.
(2S,3S,4R)-Allyl-3-((R)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-methylbut
anoyloxy)-2-(2-(4-methoxybenzyloxy)ethyl)-4-methyldecanoate (12b)
The titled compound 12b was obtained following the procedure described for 12a.
Flash column chromatography (petroleum ether: ethyl acetate = 9:1); yield: 92%;
colorless oil; [α]20 = − 43.1 (c = 1.0, CHCl3). νmax(KBr): 3443, 3377, 2952, 2931,
D
2860, 1740, 1734, 1612, 1512, 1463, 1366, 1248, 1173, 1092, 1038, 986, 931, 820
cmꢀ1; 1H NMR (400 MHz, CDCl3) δ 7.67 (d, J = 7.5 Hz, 2H), 7.52 (d, J = 7.2 Hz, 2H),
7.30 (t, J = 7.3 Hz, 2H), 7.23 (t, J = 7.2 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H), 6.77 (d, J =
8.5 Hz, 2H), 5.83 – 5.66 (m, 1H), 5.24 – 5.17 (m, 1H), 5.17 – 5.10 (m, 2H), 5.08 (d,
J= 10.3 Hz, 1H), 4.41 (dd, J = 13.4, 5.7 Hz, 2H), 4.33 – 4.20 (m, 5H), 4.14 (t, J = 7.0
Hz, 1H), 3.69 (s, 3H), 3.42 – 3.26 (m, 2H), 2.96 – 2.79 (m, 1H), 2.13 (dt, J = 20.3, 6.8
Hz, 1H), 1.92 – 1.81 (m, 1H), 1.72 – 1.62 (m, 2H), 1.34 (s, 1H), 1.27 – 1.09 (m, 10H),
0.91 (d, J = 6.7 Hz, 3H), 0.85 – 0.69 (m, 11H).13C NMR (100 MHz, CDCl3) δ 172.6,
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171.3, 159.2, 156.2, 144.0, 143.9, 141.3, 132.0, 130.3, 129.3, 127.7, 127.1, 125.2,
125.1, 120.0, 118.7, 113.8, 77.9, 72.8, 67.3, 67.0, 65.5, 59.1, 55.3, 47.2, 45.3, 34.5,
33.7, 31.8, 31.1, 29.4, 29.2, 27.1, 27.0, 22.7, 19.5, 17.1, 14.1, 13.8. HRMS–MALDI
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(m/z): [M + Na]+ calcd for C44H57NNaO8 , 750.3976, found 750.3973.
(R,E)-Allyl-4-((5R,8S,11S,12S)-1-(9H-fluoren-9-yl)-8-isopropyl-12-(2-(4-methoxybe
nzyloxy)ethyl)-5-methyl-11-((R)-octan-2-yl)-3,6,9-trioxo-2,10-dioxa-4,7-diazatrideca
namido)-5-(tert-butyldimethylsilyloxy)pent-2-enoate (13a)
The ester 12a (3.63 g, 4.99 mmol) was dissolved in CH2Cl2 (30 mL) and diethylamine
(15 mL) was added. After 2 h, the solvent was removed and the residue was purified
by column chromatography on silica gel (petroleum ether: ethyl acetate = 10:1 to 2:1)
to afford the amino ester as colorless oil.
The obtained amine (1.61 g, 3.20 mmol) and Fmoc amino acid 5a (1.20 g, 3.84 mmol)
was dissolved in anhydrous CH2Cl2 (30 mL). And HOBt (520 mg, 3.84 mmol), EDCI
(740 mg, 3.84 mmol), Et3N (535 ꢁL, 3.84 mmol) was added successively. The
reaction mixture was stirred for 18 h and the solvent was removed. The residue was
dissolved in CH2Cl2 (100 mL) and washed successively with 1% HCl, saturated
aqueous NaHCO3, brine, dried over Na2SO4, filtrated and concentrated under reduced
pressure. The residue was purified by flash chromatography on silica gel (petroleum
ether: ethyl acetate = 10: 1 to 5:1) to obtain the amide as a colorless oil.
To a solution of obtained amide (2.30 g, 2.88 mmol) in anhydrous THF (30 mL),
Pd(PPh3)4 (666 mg, 0.576 mmol) and Nꢀmethylaniline (625 ꢁL, 5.76 mmol) were
added. The reaction mixture was stirred for 1 h at room temperature, and diluted with
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ethyl acetate (200 mL). The organic phase was washed by 1% HCl (2 × 60 mL), dried
over Na2SO4 and concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel (petroleum ether: ethyl acetate = 20: 1 to 1: 1) to
afford the acid.
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The obtained acid above and amine 3a (1.05 g, 1.38 mmol) was dissolved in
anhydrous CH2Cl2 (14 mL), and TEA (230 ꢁL, 1.66 mmol), HOBt (224 mg, 1.66
mmol) and EDCI (318 mg, 1.66 mmol) were added successively. The reaction mixture
was stirred for 18 h and the solvent was removed. The residue was diluted with
CH2Cl2 (100 mL) and washed successively with 1 % HCl, saturated aqueous NaHCO3,
brine, dried over Na2SO4 and filtrated. The filtrate was concentrated under reduced
pressure. The residue was purified by column chromatography on silica gel
(petroleum ether: ethyl acetate = 5: 1) to obtain compound 13a (1.04 g, 20 % for 4
steps) as a colorless oil. [α]20D = − 8.5 (c = 0.8, CHCl3); νmax(KBr): 3322, 2931, 2860,
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1726, 1665, 1515, 1364, 1181, 1105, 1039, 989, 935, 839 cmꢀ1; H NMR (400 MHz,
CDCl3) δ 7.74 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 7.1 Hz, 2H), 7.38 (t, J = 7.4 Hz, 2H),
7.27 (dd, J = 13.1, 5.8 Hz, 2H), 7.19 (d, J = 7.8 Hz, 2H), 6.92 – 6.79 (m, 4H), 6.23 (d,
J = 7.8 Hz, 1H), 6.09 (d, J = 7.7 Hz, 1H), 5.96 – 5.80 (m, 2H), 5.28 (t, J = 14.1 Hz,
1H), 5.19 (d, J = 10.2 Hz, 1H), 5.08 (t, J = 5.4 Hz, 1H), 4.70 – 4.61 (m, 1H), 4.57 (d,
J = 5.4 Hz, 2H), 4.48 – 4.26 (m, 6H), 4.20 (t, J = 7.0 Hz, 1H), 3.76 (s, 2H), 3.69 –
3.63 (m, 1H), 3.63 – 3.57 (m, 1H), 3.51 – 3.44 (m, 1H), 3.43 – 3.35 (m, 1H), 2.79 (dd,
J = 13.8, 6.0 Hz, 1H), 2.13 – 2.01 (m, 1H), 1.83 – 1.66 (m, 3H), 1.44 – 1.34 (m, 5H),
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1.34 – 1.16 (m, 13H), 1.18 – 1.06 (m, 2H), 0.97 – 0.78 (m, 28H), 0.03 (s, 6H). C
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NMR (100 MHz, CDCl3) δ 171.3, 170.7, 169.2, 164.4, 158.2, 155.1, 144.9, 142.8,
140.3, 131.0, 129.0, 128.3, 126.7, 126.1, 124.1, 124.1, 121.0, 118.9, 117.2, 112.8,
71.6, 66.1, 65.8, 64.1, 63.3, 57.1, 54.2, 50.6, 49.5, 46.1, 44.8, 33.8, 32.7, 30.7, 30.1,
28.6, 28.5, 25.7, 24.8, 21.6, 18.3, 17.8, 17.3, 16.7, 13.4, 13.1, ꢀ6.5. HRMS–MALDI
(m/z): [M + Na]+ calcd for C58H83N3NaO11Si+, 1048.5689, found 1048.5695.
(S,E)-Allyl-4-((5S,8R,11S,12S)-1-(9H-fluoren-9-yl)-8-isopropyl-12-(2-(4-methoxybe
nzyloxy)ethyl)-5-methyl-11-((R)-octan-2-yl)-3,6,9-trioxo-2,10-dioxa-4,7-diazatrideca
namido)-5-(tert-butyldimethylsilyloxy)pent-2-enoate (13b)
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The titled compound 13b was obtained following the general procedure described for
13a. Flash column chromatography (petroleum ether: ethyl acetate = 5:1); yield, 21%
for 4 steps; colorless oil;[α]20 = − 23.8 (c = 1.74, CHCl3); νmax(KBr): 3322, 3041,
D
2931, 2860, 2741, 1727, 1665, 1614, 1515, 1460, 1364, 1515, 1460, 1364, 1302, 1251,
1181, 1105, 1039, 989, 839 cmꢀ1; 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.5 Hz,
2H), 7.61 (d, J = 7.1 Hz, 2H), 7.38 (t, J = 7.5 Hz, 2H), 7.31 (dd, J = 13.8, 6.7 Hz, 2H),
7.07 – 6.88 (m, 4H), 6.86 – 6.65 (m, 3H), 6.11 (d, J = 15.7 Hz, 1H), 5.97 – 5.83 (m,
2H), 5.27 (dd, J = 17.3, 1.3 Hz, 1H), 5.19 (dd, J = 10.4, 1.0 Hz, 1H), 4.97 (s, 1H),
4.76 (s, 1H), 4.66 – 4.59 (m, 2H), 4.55 – 4.45 (m, 2H), 4.33 – 4.08 (m, 5H), 3.79 –
3.68 (m, 5H), 3.38 – 3.30 (m, 1H), 3.27 – 3.19 (m, 1H), 2.19 – 2.08 (m, 1H), 2.03 (s,
1H), 1.42 (s, 4H), 1.31 – 1.20 (m, 14H), 0.99 – 0.83 (m, 30H), 0.06 (s, 6H). 13C NMR
(100 MHz, CDCl3) δ 172.8, 171.4, 171.3, 165.8, 159.2, 156.7, 146.6, 143.9, 141.4,
132.2, 129.6, 127.8, 127.6, 127.1, 127.1, 125.0, 124.9, 121.5, 120.0, 119.9, 118.1,
113.6, 78.5, 72.9, 67.6, 65.0, 64.4, 58.3, 55.2, 51.7, 47.1, 46.4, 38.6, 34.8, 33.4, 31.8,
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