Total Synthesis of the Antitumor Depsipeptide FE399 and Its S-Benzyl Derivative: A Macrolactamization Approach

Article abstract of DOI:10.1002/ejoc.202000459

An efficient and practical method for the synthesis of (9R,14R,17R)-FE399, a novel antitumor bicyclic depsipeptide, was developed. A 2-methyl-6-nitrobenzoic anhydride (MNBA)-mediated dehydration condensation reaction was effectively employed for the formation of the 16-membered macrocyclic depsipeptide moiety of FE399. FE399 was found to exist as an inseparable equilibrium mixture of conformational isomers; the mixture was quantitatively transformed into the corresponding S-benzyl product and isolated as a single isomer. Thus, we could confirm that the molecular structure of FE399 obtained by this method is identical to that of the natural product.

Full text of DOI:10.1002/ejoc.202000459

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: Total Synthesis of the Antitumor Depsipeptide FE399 and its S-
Benzyl Derivative:
A Macrolactamization Approach
Authors: Takayuki Tonoi, Miyuki Ikeda, Teruyuki Sato, Takehiko
Inohana, Ryo Kawahara, Takatsugu Murata, and Isamu
Shiina
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202000459
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01/2020

10.1002/ejoc.202000459
European Journal of Organic Chemistry
Total Synthesis of the Antitumor Depsipeptide FE399 and its S-Benzyl Derivative:
A Macrolactamization Approach
Takayuki Tonoi,* Miyuki Ikeda, Teruyuki Sato, Takehiko Inohana, Ryo Kawahara, Takatsugu Murata,
Isamu Shiina*
Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka,
Shinjuku-ku, Tokyo 162-8601, Japan
*Corresponding authors: tonoi@rs.tus.ac.jp (T. Tonoi), shiina@rs.tus.ac.jp (I. Shiina).
Abstract: An efficient and practical method for the synthesis of (9R,14R,17R)-FE399, a novel antitumor
bicyclic depsipeptide, was developed. A 2-methyl-6-nitrobenzoic anhydride (MNBA)-mediated dehydration
condensation reaction was effectively employed for the formation of the 16-membered macrocyclic
depsipeptide moiety of FE399. FE399 was found to exist as an inseparable equilibrium mixture of
conformational isomers; the mixture was quantitatively transformed into the corresponding S-benzyl product
and isolated as a single isomer. Thus, we could confirm that the molecular structure of FE399 obtained by
this method is identical to that of the natural product.
INTRODUCTION
A novel bicyclic depsipeptide, FE399 (1), was first isolated from the filamentous fungus, Ascochyta sp.,
by Oobayashi et al. of the Ajinomoto group1 (see Figure 1). During the screening process of antitumor
agents as apoptosis-inducing lead compounds, particularly on colorectal cancer cells with p53 mutations, it
was found that the compound 1 exhibits the antitumor activity via the induction of apoptosis of a human
colorectal cancer cell in vitro, particularly against mutant p53 but not against the wild-type p53 in the broth
culture of the fungus.1
Most human cancer cells generally develop due to genetic mutations, after which they proliferate
indefinitely without apoptosis. Generally, gene mutations are caused by various factors such as aging,
ultraviolet light, and mutagenic compounds.2 Among such mutated genes, mutant p53 is frequently found in
malignant human tumors.3 The p53 tumor protein undergoes a broad range of cellular processes, including
metabolism, cycle arrest, and apoptosis, that maintain cell homeostasis.3 Once p53 has undergone mutation
and is inactive for some reason, the probability of the emergence of tumor cells increases. Therefore, this
compound is a novel and promising candidate for use in anticancer drugs.
1
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10.1002/ejoc.202000459
European Journal of Organic Chemistry
Figure 1. Structure of FE399 (1)
Contrarily, compound 1 was determined to have a molecular structure consisting of a 16-membered
bicyclic depsipeptide comprising a -hydroxy fatty acid moiety and two cysteine molecules bridged by an
8-membered cyclic disulfide.1,4,5 However, thus far, the relative and absolute configurations at the C9, C14,
and C17 chiral centers of 1 as well as its specific optical rotation have not been revealed. Moreover,
compound 1 is found to exist as a mixture of conformational isomers, presumably due to the preferred
conformations of the strained bicyclic ring system containing a disulfide-bridged peptide moiety; this
explains why it was difficult for the Ajinomoto group to isolate FE399 as a single compound. Therefore,
despite its promising pharmacological effects, the advancement of the biological studies on FE399 has been
hampered. Thus, this study is aimed at the total synthesis of FE399 to uncover the absolute stereostructure of
the natural product and to advance its biological activity study.6
First, we synthetically determined the planar structure of FE399. Among all the stereoisomers of 1, we
first attempted to synthesize (9R,14R,17R)-1 comprising two residues of a readily available natural L-cystein
while, more recently, Katsuta et al. revealed that the relative stereochemistry of FE399 is most likely to be
the (9R,14R,17R)-form.6 The retrosynthetic analysis of (9R,14R,17R)-1 is shown in Scheme 1. We envisage
that macrolactamization mediated by 2-methyl-6-nitrobenzoic anhydride (MNBA)7a-d, followed by
intramolecular disulfide bond formation would be suitable for the construction of the depsipeptide backbone
of the target molecule 1 without the epimerization of the C14 and C17 stereogenic centers. In fact, although
the recently established method using the combination of MNBA and 4-(dimethylamino)pyridine N-oxide
(DMAPO) as a weaker base than 4-(dimethylamino)pyridine (DMAP) was found to be effective in the
synthesis of amides and peptides,7e,f a macrolactamization approach using MNBA for the total synthesis of
natural products has not been reported so far.
The MNBA-mediated dehydration condensation reaction of the corresponding precursor 3 could be
employed to form the 16-membered depsipeptide core of 2. The cyclization precursor 3 could be linearly
synthesized via the successive esterification and amidation of a -hydroxy fatty acid ester 4 with the
commercially available cysteine derivative, Fmoc-Cys(Trt)-OH. The fatty acid ester 4 could be efficiently
constructed through Forsyth’s methodology8 on one-pot operation for the synthesis of epoxide derived from
a known diol 59 and nucleophilic opening of the epoxide with Grignard reagent 6.10
2
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10.1002/ejoc.202000459
European Journal of Organic Chemistry
Scheme 1. Retrosynthetic analysis of (9R,14R,17R)-FE399 (1)
The synthetic route to the desired compound 3 as a cyclization precursor is shown in Scheme 2. First, the
known vicinal diol 5 derived from D-norvaline was subjected to stereoselective epoxidation using
N-(2,4,6-triisopropylbenzenesufonyl)imidazole (N-TrisIm) and NaH, and a nucleophilic epoxide
ring-opening reaction with the presynthesized Grignard reagent 610 in the presence of a catalytic amount of
CuI and 2,2’-bipyridyl (bpy) to afford the desired secondary alcohol 7 in a one-pot operation. This was
followed by the p-methoxybenzyl (PMB) protection of the secondary hydroxyl group in 7 and the
deprotection of the triisopropylsilyl (TIPS) group to afford the primary alcohol 9 in 77% yield (4 steps).
After the subsequent oxidation of 9, the obtained aldehyde 10 was further oxidized to the corresponding
carboxylic acid 11 followed by the allyl ester protection of the carboxyl group in 11 and the deprotection of
the PMB group in 12 to afford the desired ester 4 in excellent yields.
With the desired -hydroxy fatty acid ester moiety in hand, next, the depsipeptide moiety of 3 comprising
two cysteine residues was constructed. We envisioned that the iterative ligations of two cysteines would be
suitable for the synthesis of 3, thereby effectively preventing racemization at the -carbon atom of each
amino acid residue. The MNBA-mediated esterification of 4 with Fmoc-Cys(Trt)-OH in the presence of
4-(dimethylamino)pyridine (DMAP) as a nucleophilic catalyst was performed to afford the ester 13 without
racemization, followed by the removal of the Fmoc group with dicyclohexylamine to yield 14 in 92% yield
(2 steps). The iterative coupling of 14 with Fmoc-Cys(Trt)-OH using MNBA with DMAP and the removal
of the Fmoc group was executed to afford the desired coupling product 16 in moderate yield (70%, 2 steps).
Eventually, the cleavage of the allyl ester 16 yielded the target molecule 3 in high yield (96%).
3
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10.1002/ejoc.202000459
European Journal of Organic Chemistry
Scheme 2. Synthesis of the cyclization precursor 3.
Subsequently, the macrolactamization of the obtained cyclization precursor 3 was attempted using the
MNBA method in the presence of DMAP or DMAPO as a nucleophilic catalyst (Table 1). When a slight
amount of MNBA (1.3 equiv.) and an excess amount of DMAP (6.0 equiv.) were employed in a dilute
solution of dichloromethane (2 mM) at room temperature, the desired macrolactam 2 was obtained in
satisfactory yield (entry 1, 66%). Increasing the reaction temperature to 40°C improved the yield (74%,
entry 2), whereas in the presence of excess triethylamine (Et3N) (3.0 equiv.) as a co-base, decreasing the
amount of DMAP to 0.2 equiv. reduced the yield (45%, entry 3). Moreover, the combination of MNBA and
DMAPO was effective as coupling agents in this reaction (entries 4 and 5). In particular, with the use of a
catalytic amount of DMAPO (0.2 equiv.) in the presence of Et3N (3.0 equiv.), the macrolactamization
proceeded and afforded products in good yield (77%). Conversely, when a combination of HATU (1.3
equiv.) and diisopropylethylamine (2.6 equiv.), one of the conventionally used coupling reagents for peptide
4
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10.1002/ejoc.202000459
European Journal of Organic Chemistry
bond formation, was employed, the reaction afforded a low product yield (entry 6, 38%).
Table 1. Synthesis of macrolactam 2.
Having established an effective method for the construction of the desired 16-membered macrolactam, we
executed the final step in the synthesis of FE399. As shown in Scheme 3, oxidative disulfide bond formation
using iodine of the cyclized product 2 provided the target compound (9R,14R,17R)-1 in moderate yield as a
mixture of conformational isomers because of its strained bicyclic ring system. Although the separation of
each isomer of (9R,14R,17R)-1 was attempted using preparative thin-layer chromatography (TLC), no
isomer was successfully isolated because these conformers were in dynamic equilibrium with each other.
After the disulfide bond formation process and the subsequent preparative TLC separation, mainly three
fractions were obtained, corresponding to the desired products that appeared as spots on the plate. In fact,
each mass spectrum (HRMS) of the three fractions proved to be identical to that of FE399. Moreover, the
carbon nuclear magnetic resonance (13C NMR) chemical shifts were assigned to the major isomer of the
mixtures obtained, and they were in complete agreement with the literature data1,4 reported for the major
component of the inseparable mixture of FE399 (Table 2).11
Scheme 3. Transformation of 2 into the S-benzyl product 19 via the formation of FE399.
5
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European Journal of Organic Chemistry
Therefore, we further attempted to transform the obtained mixture of (9R,14R,17R)-1 into the
corresponding S-alkylated product, through which we expected that the target product would be isolated as a
single isomer. Eventually, the alkylation of the mixture of (9R,14R,17R)-1 with benzyl bromide was
performed in the presence of tris(2-carboxyethyl)phosphine hydrochloride (TCEPꞏHCl) as a reducing agent
to cleave the disulfide bond and afford the corresponding S-benzyl product 17 quantitatively (Scheme 3).
Notably, the results not only provided indirect evidence of the formation of (9R,14R,17R)-1 but also
indicated that the structure of the synthetic product, (9R,14R,17R)-1, would be the most likely relative
configuration of the natural FE399.
Table 2. 13C NMR shifts (ppm) of the natural and synthetic products in DMSO-d6
In conclusion, the total synthesis of (9R,14R,17R)-FE399 (1) was efficiently achieved through a
macrolactamization approach mainly by exploiting the MNBA-mediated dehydration condensation reaction
for the construction of the 16-membered depsipeptide core of FE399 using a known diol (5) in 15 steps, with
an overall yield of 20%. It is noteworthy that this is the first application of the MNBA-mediated
macrolactamization for the total synthesis of natural products. Further studies to clarify the structures of the
depsipeptides related to FE399 and to evaluate their biological activities are underway.
6
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10.1002/ejoc.202000459
European Journal of Organic Chemistry
EXPERIMENTAL SECTION
General information. Optical rotations were determined using a Jasco P-1020 polarimeter. Infrared (IR)
spectra were obtained using a Horiba FT-330 or Jasco FT/IR-4600 Fourier transform infrared spectrometer.
13
Proton and carbon nuclear magnetic resonance (1H and C NMR) spectra were recorded on the following
instruments: JEOL JNM-AL500 (1H at 500 MHz and 13C at 125 MHz). Mass spectra were determined by a
Bruker Daltonics micrOTOF focus (ESI-TOF) mass spectrometer. Thin layer chromatography was
performed on Wakogel B5F. HPLC was performed with a Hitachi LaChrom Elite system composed of the
Organizer, L-2400 UV Detector, and L-2130 Pump. All reactions were carried out under argon atmosphere
in dried glassware unless otherwise noted. CH2Cl2 was distilled from diphosphorus pentoxide, then calcium
hydride, and dried over MS4A, toluene was distilled from diphosphorus pentoxide, and dried over MS4A,
and THF and diethyl ether were distilled from sodium/benzophenone immediately prior to use. All reagents
were purchased from Tokyo Kasei Kogyo Co., Ltd., Kanto Chemical Co., Inc. or Aldrich Chemical Co., Inc.,
and used without further purification unless otherwise noted. MNBA was purchased from Tokyo Kasei
Kogyo Co. Ltd. (TCI M1439).
Experimental procedures and analytical data.
(R)-9-(4-Methoxybenzyloxy)dodecan-1-ol (9)
A 55% dispersion of NaH in paraffinni liquid (242 mg, 5.6 mmol) was added to a stirred solution of diol 5
(208 mg, 2.0 mmol) in tetrahydrofuran (19.9 mL) at 0 ºC. After warming to room temperature, stirring was
continued
for
1
h.
The
reaction
mixture
was
cooled
to
0
ºC
and
N-(2,4,6-triisopropylbenzenesulfonyl)imidazole (733 mg, 2.2 mmol) was added. The mixture was stirred at
room temperature for 2 h, followed by the addition of CuI (76 mg, 0.40 mmol) and 2,2’-bipyridyl (62 mg,
0.40 mmol) at 40 ºC. After stirring for 5 min, a solution of Grignard reagent 6 (0.31 M in THF) prepared in
advance was added dropwise into the above mixture, which was stirred at 40 ºC for 1 h and at 0 ºC for
another 1 h. After cooling to 40 ºC , the reaction mixture was quenched with saturated aqueous ammonium
chloride. The organic layer was separated and the aqueous layer was extracted with EtOAc. The combined
organic layer was dried over sodium sulfate. After evaporation of the solvent, the crude product was purified
by column chromatography on silica gel (hexane/EtOAc = 20/1 to 10/1) to afford 7 accompanied by an
inseparable and unidentified by-product, which was used in the next step without further purification.
To a cooled (0 ºC) solution of alcohol 7 in DMF (17 mL) was added potassium tert-butoxide (1.0 M in
tetrahydrofuran, 3.4 mL, 3.4 mmol). After the solution was stirred for 30 min, 4-methoxybenzyl chloride
(0.74 mL, 5.5 mmol) and tetrabutylammonium Iodide (124 mg, 0.34 mmol) was successively added. After
warming to room temperature and stirringfor 20 h, the reaction mixture was quenched with saturated
aqueous ammonium chloride. The organic layer was separated and the aqueous layer was extracted with
7
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10.1002/ejoc.202000459
European Journal of Organic Chemistry
diethyl ether. The combined organic layer was dried over sodium sulfate. After evaporation of the solvent,
the crude product was purified by column chromatography on silica gel (hexane/EtOAc = 20/1) to afford 8
along with a small amount of inseparable by-products, which was used in the next step without further
purification.
To a cooled (0 ºC) solution of ether 8 in tetrahydrofuran (17 mL) was added tetrabutylammonium fluoride
(1.0 M in tetrahydrofuran, 5.0 mL, 5.0 mmol), and the solution was stirred at room temperature for 4 h. The
reaction was then quenched with saturated aqueous sodium hydrogencarbonate. The organic layer was
separated and the aqueous layer was extracted with EtOAc. The combined organic layer was washed with
brine, and dried over sodium sulfate. After evaporation of the solvent, the crude product was purified by
column chromatography on silica gel (hexane/EtOAc = 10/1 to 4/1), followed by purification through thin
layer chromatography on silica gel (hexane/EtOAc = 5/1) to afford alcohol 9 as a colorless oil (492 mg, 77%,
95% ee).
[α]D26 0.39 (c 1.00, CHCl3)
1
IR (neat) 3402, 2931, 2854 cm
HPLC analysis DAICEL CHIRALPAC IA-3, UV 254 nm, temperature 25 °C, hexane/iPrOH = 99/1, flow
rate 1.0 mL/min, tR = 33.5 min, 40.2 min
1H NMR (500 MHz, CDCl3) δ 7.29–7.26 (m, 2H, PMB), 6.89–6.85 (m, 2H, PMB), 4.43 (s, 2H, PMB), 3.80
(s, 1H, PMB), 3.64 (t, J = 6.5 Hz, 2H, 1-H), 3.37–3.32 (m, 1H, 9-H), 1.60–1.25 (m, 18H, 2-H, 3-H, 4-H, 5-H,
6-H, 7-H, 8-H, 10-H, 11-H), 0.90 (t, J = 7.0 Hz, 3H, 12-H).
13C NMR (125 MHz, CDCl3)159.0, 131.3, 129.2, 113.6, 78.4, 70.3, 62.9, 55.2, 36.1, 33.8, 32.7, 29.7, 29.5,
29.3, 25.7, 25.3, 18.6, 14.3
HRMS (ESI/TOF) m/z [M+Na]+ cacld for C20H34O3Na 345.2400, found 345.2393.
(R)-9-(4-Methoxybenzyloxy)dodecanal (10)
To a solution of 9 (319 mg, 0.99 mmol) in dichloromethane (7.9 mL) and dimethyl sulfoxide (2.0 mL) were
added triethylamine (2.6 mL, 19 mmol) and SO3·pyridine complex (1.48 g, 9.3 mmol) at 0 ºC. After the
solution was stirred at room temperature for 2 h, the reaction mixture was quenched with saturated aqueous
ammonium chloride. The organic layer was separated and the aqueous layer was extracted with
dichloromethane. The combined organic layer was washed with brine, and dried over sodium sulfate. After
evaporation of the solvent, the crude product was purified by column chromatography on silica gel
(hexane/EtOAc = 20/1 to 10/1) to afford aldehyde 10 as a colorless oil (293 mg, 92%).
[α]D23 +2.04 (c 0.67, CHCl3)
1
IR (neat) 2931, 2862, 1728 cm
1H NMR (500 MHz, CDCl3) δ 9.77 (t, J = 2.0 Hz, 1H, 1-H), 7.28–7.24 (m, 2H, PMB), 6.89–6.85 (m, 2H,
8
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10.1002/ejoc.202000459
European Journal of Organic Chemistry
PMB), 4.43 (s, 2H, PMB), 3.80 (s, 3H, PMB), 3.37–3.32 (m, 1H, 9-H), 2.42 (td, J = 7.5, 2.0 Hz, 2H, 2-H),
1.67–1.59 (m, 2H, 3-H), 1.59–1.25 (m, 14H, 4-H, 5-H, 6-H, 7-H, 8-H, 10-H, 11-H), 0.91 (t, J = 7.5 Hz, 3H,
12-H).
13C NMR (125 MHz, CDCl3)202.8, 159.0, 131.2, 129.2, 113.6, 78.4, 70.3, 55.2, 43.8, 36.1, 33.8, 29.5,
29.3, 29.1, 25.2, 22.0, 18.6, 14.2.
HRMS (ESI/TOF) m/z [M+Na]+ cacld for C20H32O3Na 343.2244, found 343.2244.
Allyl (R)-9-(4-methoxybenzyloxy)dodecanoate (12)
To a solution of aldehyde 10 (1.02 mg, 3.2 mmol) in tetrahydrofuran (21 mL), tert-butyl alcohol (21 mL)
and 2-methyl-2-butene (1.4 mL, 13 mmol) was added, followed by the addition of a solution of NaH2PO4
(571 mg, 4.8 mmol) and NaClO2 (1.1 g, 9.6 mmol) in H2O (21.3 mL). After the reaction mixture was stirred
at room temperature for 4.5 h, the organic layer was separated and the aqueous layer was extracted with
diethyl ether. The combined organic layer was washed with brine, and dried over sodium sulfate. After
evaporation of the solvent, the crude product containing carboxylic acid 11 was used in the next step without
further purification. To a solution of 11 dissolved in acetone (20 mL) was added Cs2CO3 (1.2 g, 3.5 mmol).
After the dispersion was stirred for 10 min, a solution of allyl bromide (425 mg, 1.0 mmol) in acetone (12
mL) was added, and the mixture was warmed to 50 °C. After stirring for 1 h, the reaction mixture was
cooled to room temperature and filtrated through a pad of Celite®. After removing solvent in vacuo, the
crude product was purified by column chromatography on silica gel (hexane/EtOAc = 20/1) to afford 12 as a
colorless oil (1.1 mg, 93%).
[α]D27 +0.04 (c 1.87, CHCl3)
1
IR (neat) 2931, 2862, 1736 cm
1H NMR (500 MHz, CDCl3) δ 7.28–7.24 (m, 2H, PMB), 6.89–6.85 (m, 2H, PMB), 5.92 (ddt, J = 17.0 Hz,
10.5 Hz, 6.0 Hz, 1H, Allyl), 5.31 (ddd, J = 17.0 Hz, 3.0 Hz, 1.5 Hz, 1H, Allyl), 5.23 (ddd, J = 10.5 Hz, 3.0
Hz, 1.5 Hz, 1H, Allyl), 4.58 (dt, J = 6.0 Hz, 1.5 Hz, 2H, Allyl), 4.42 (s, 2H, PMB), 3.80 (s, 3H, PMB), 3.37–
3.31 (m, 1H, 9-H), 2.33 (t, J = 7.5 Hz, 2H, 2-H), 1.63 (q, J = 7.5 Hz, 2H, 3-H), 1.55–1.23 (m, 14H, 4-H, 5-H,
6-H, 7-H, 8-H, 10-H, 11-H), 0.90 (t, J = 7.5 Hz, 3H, 12-H)
13C NMR (125 MHz, CDCl3) 173.5, 159.0, 132.3, 131.3, 129.2, 118.0, 113.7, 78.4, 70.4, 64.9, 55.2, 36.2,
34.2, 33.8, 29.6, 29.2, 29.1, 25.3, 24.9, 18.6, 14.3
HRMS (ESI/TOF) m/z [M+Na]+ cacld for C23H36O4Na 399.2506, found 399.2501.
Allyl (R)-9-hydroxydodecanoate (4)
9
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10.1002/ejoc.202000459
European Journal of Organic Chemistry
To a cooled (0 ºC) solution of 12 (285 mg, 0.76 mmol) in dichloromethane (13.8 mL) and phosphate buffer
solution (1.4 mL) were added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (206 mg,0.91 mmol). After the
reaction mixture was stirred at room temperature for 2 h, the reaction was quenched with saturated aqueous
sodium hydrogencarbonate. The organic layer was separated and the aqueous layer was extracted with
dichloromethane. The combined organic layer was washed with brine, and dried over sodium sulfate. After
evaporation of the solvent, the crude product was purified by column chromatography on silica gel
(hexane/diethyl ether = 5/1 to 3/1) to afford 4 as a colorless oil.(184 mg, 95%).
[α]D24 1.07 (c 0.88, CHCl3)
1
IR (neat) 3425, 2923, 2862, 1728 cm
1H NMR (500 MHz, CDCl3) δ 5.92 (ddt, J = 17.0 Hz, 10.0 Hz; 6.0 Hz, 1H, Allyl), 5.31 (ddd, J = 17.0 Hz,
3.0 Hz, 1.5 Hz, 1H, Allyl), 5.23 (ddd, J = 10.0 Hz, 3.0 Hz, 1.5 Hz, 1H, Allyl), 4.57 (dt, J = 6.0 Hz, 1.5 Hz,
2H, Allyl), 3.63–3.56 (m, 1H, 9-H), 2.33 (t, J = 7.5 Hz, 2H, 2-H), 1.63 (quint, J = 7.5 Hz, 2H, 3-H), 1.52–
1.25 (m, 14H, 4-H, 5-H, 6-H, 7-H, 8-H, 10-H, 11-H), 0.93 (t, J = 7.0 Hz, 3H, 12-H)
13C NMR (125 MHz, CDCl3) 173.5, 132.3, 118.1, 71.6, 64.9, 39.7, 37.4, 34.2, 29.4, 29.2, 29.0, 25.5, 24.9,
18.8, 14.1
HRMS (ESI/TOF) m/z [M+Na]+ cacld for C15H28O3Na 279.1931, found 279.1938.
Allyl (R)-9-(S-trityl-L-cysteinyloxy)dodecanoate (14)
To a cooled (0 ºC) solution of 4 (282 mg, 1.10 mmol) in dichloromethane (11 mL) was added a solution of
N-(9-fluorenylmethoxycarbonyl)-S-triphenylmetyl-L-cysteine (772 mg, 1.32 mmol), MNBA (477 mg, 1.43
mmol) and DMAP (349 mg, 2.85 mmol), successively. The reaction mixture was stirred at room temperature
for 1.5 h, followed by the addition of a saturated aqueous sodium hydrogencarbonate solution. The organic
layer was separated and the aqueous layer was extracted with dichloromethane. The combined organic layer
was washed with brine, and dried over sodium sulfate. After evaporation of the solvent, the crude product
was purified by column chromatography on silica gel (chloroform to chloroform/methanol = 100/1). The
resulting product 13 obtained along with inseparable and unidentified by-products was used in the next step
without further purification. To a solution of 13 in N,N-dimethylformamide (5.1 mL) was added
dicyclohexylamine (5.1 mL). The solution was stirred at room temperature for 4 h. After evaporation of the
solvent, the crude product was purified by column chromatography on silica gel (chloroform) to afford 14 as
a pale yellow oil (606 mg, 92%).
[α]D22 34.1 (c 1.12, CHCl3).
1
IR (neat) 3438, 3386, 3062, 3024, 2924, 2854, 1728 cm
10
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10.1002/ejoc.202000459
European Journal of Organic Chemistry
1H NMR (500 MHz, CDCl3) δ 7.45–7.18 (m, 15H, Trt), 5.92 (ddt, J = 17.0, 10.5, 5.5 Hz, 1H, Allyl), 5.31
(ddd, J = 17.0, 3.0, 1.5 Hz, 1H, Allyl), 5.23 (ddd, J = 5.5, 3.0, 1.5 Hz, 1H, Allyl), 4.85–4.78 (m, 1H, 9-H),
4.57 (td, J = 5.5, 1.5 Hz, 2H, Allyl), 3.17 (dd, J = 8.5, 4.5 Hz, 1H, 2'-H), 2.51 (dd, J = 12.0, 4.5 Hz, 1H,
3'-H), 2.45 (dd, J = 12.0, 8.5 Hz, 1H, 3'-H), 2.32 (t, J = 7.5 Hz, 2H, 2-H), 1.65–1.56 (m, 2H, 3-H), 1.50–
1.39 (m, 4H, 8-H, 10-H), 1.33–1.13 (m, 10H, 4-H, 5-H, 6-H, 7-H, 11-H), 0.84 (t, J = 7.5 Hz, 3H, 12-H)
13C NMR (125 MHz, CDCl3)173.5, 173.3, 144.5, 132.2, 129.5, 127.8, 126.6, 118.0, 75.0, 66.7, 64.8, 54.0,
37.1, 36.0, 34.1, 33.8, 29.2, 29.0, 28.9, 25.0, 24.7, 18.3, 13.9
HRMS (ESI/TOF) m/z [M+H]+ cacld for C37H48NO4S 602.3286, found 602.3299.
Allyl (R)-9-(S-trityl-N-(S-trityl-L-cysteinyl)-L-cysteinyloxy)dodecanoate (16)
To a cooled (0 ºC) solution of 14 (139 mg, 0.23 mmol) in dichloromethane (2.3 mL) was added a solution of
N-(9-fluorenylmethoxycarbonyl)-S-triphenylmetyl-L-cysteine (162 mg, 0.28 mmol), MNBA (103 mg, 0.30
mmol) and DMAP (73 mg, 0.60 mmol), successively. After the reaction mixture was stirred at room
temperature for 3 h, the reaction was quenched with saturated aqueous sodium hydrogencarbonate. The
organic layer was separated and the aqueous layer was extracted with dichloromethane. The combined
organic layer was washed with brine, and dried over sodium sulfate. After evaporation of the solvent, the
crude product was purified by column chromatography on silica gel (chloroform/methanol = 100/1 to 10/3).
The resulting product 15 obtained along with inseparable and unidentified by-products was used in the next
step without further purification. To a solution of 15 in N,N-dimethylformamide (1.0 mL) was added
dicyclohexylamine (1.0 mL).The solution was stirred at room temperature for 4 h. After evaporation of the
solvent, the crude product was purified by column chromatography on silica gel (chloroform) to afford 16 as
a pale yellow oil (153 mg, 70% (2 steps)).
[α]D25 13.7 (c 1.83, CHCl3)
1
IR (neat) 3379, 3062, 3024, 2931, 2862, 1736, 1674 cm
1H NMR (500 MHz, CDCl3) δ 7.66 (d, J = 8.0 Hz, 1H, NH), 7.56–7.17 (m, 30H, Trt), 5.93 (ddt, J = 17.0,
11.0, 6.0 Hz, 1H, Allyl), 5.33 (dd, J = 17.0, 1.0 Hz, 1H, Allyl), 5.24 (dd, J = 11.0, 1.0 Hz, 1H, Allyl), 4.89–
4.83 (m, 1H, 9-H), 4.64–4.57 (m 2H, Allyl), 4.54–4.48 (m, 1H, 2'-H), 2.99 (dd, J = 9.5, 3.5 Hz, 1H, 2''-H),
2.81 (dd, J = 13.0, 3.5 Hz, 1H, 3''-H), 2.61–2.54 (m, 2H, 3'-H, 3''-H), 2.48 (dd, J = 12.0, 4.5 Hz, 1H, 3'-H),
2.34 (t, J = 7.0 Hz, 2H, 2-H), 1.71–1.55 (m, 2H, 3-H), 1.55–1.40 (m, 4H, 8-H, 10-H), 1.38–1.15 (m, 10H,
4-H, 5-H, 6-H, 7-H, 11-H), 0.87 (t, J = 7.0 Hz, 3H, 12-H)
13C NMR (125 MHz, CDCl3) 173.3, 172.4, 169.9, 144.5, 144.2, 132.2, 129.5, 129.3, 127.9, 127.8, 126.7,
126.6, 117.9, 75.8, 66.9, 66.4, 64.8, 53.8, 50.7, 37.1, 35.9, 34.1, 33.8, 29.1, 28.9, 28.9, 25.0, 24.7, 18.3, 13.9
11
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10.1002/ejoc.202000459
European Journal of Organic Chemistry
HRMS (ESI/TOF) m/z [M+H]+ cacld for C59H67N2O5S2 947.4486, found 947.4440.
(R)-9-(S-Trityl-N-(S-trityl-L-cysteinyl)-L-cysteinyloxy)dodecanoic acid (3)
To a solution of tetrakis(triphenylphosphine)palladium (0) (5.3 mg, 5.0 μmol) in methanol (2.5 mL) and
piperidine (10 μL, 0.105 mmol) was added a solution of 16 (47 mg, 0.050 mmol) in dichloromethane (2.5
mL). The solution was stirred at room temperature for 1 h. After filtration through a pad of Celite®, the
filtrate was concentrated in vacuo. The crude product was purified by thin layer chromatography on silica
gel (chloroform/methanol = 10/1) to afford 3 as a pale yellow oil (43.3 mg, 96%), which was used in the
next cyclization reaction as soon as possible because the compound 3 obtained was often unstable with time.
1H NMR (500 MHz, CDCl3) δ 7.68 (d, J = 7.5 Hz, 1H, NH), 7.48–7.14 (m, 30H, Trt), 4.87–4.78 (m, 1H,
9-H), 4.55–4.46 (m, 1H, 2'-H), 4.01–3.56 (br, 3H, OH, NH2), 3.01–2.94 (m, 1H, 2''-H), 2.76 (dd, J = 13.0,
4.5 Hz, 1H, 3''-H), 2.69–2.49 (m, 2H, 3'-H, 3''-H), 2.42 (dd, J = 12.5, 5.0 Hz, 1H, 3'-H), 2.37–2.21 (m 2H,
2-H), 1.62–1.51 (m, 2H, 3-H), 1.51–1.35 (m, 4H, 8-H, 10-H), 1.35–1.10 (m, 10H, 4-H, 5-H, 6-H, 7-H, 11-H),
0.83 (t, J = 7.0 Hz, 3H, 12-H).
15
13
3'
1'
2'
16
12
10
14
6
O
O1
11
O
2
H
N
7
3
4
8
9
1''
5
N
H
TrtS
1'''
O
TrtS
(3R,6R,16R)-3,6-Bis(tritylsulfanylmethyl)-16-propyl-1-oxa-4,7-diazacyclohexadecane-2,5,8-trione (2)
To a solution of MNBA (11 mg, 32 μmol), DMAPO (0.7 mg, 4.9 μmol), and Et3N (8.9 μL, 64 μmol) in
dichloromethane (9.8 mL) at 40 ºC were slowly added a solution of 3 (22.2 mg, 25 μmol) in
dichloromethane (2.4 mL) with a mechanically driven syringe pump over a 12 h-period. The reaction
mixture was stirred for another 1 h, saturated aqueous sodium hydrogencarbonate was added. The organic
layer was separated and the aqueous layer was extracted with dichloromethane, and the organic layer was
washed with brine, dried over sodium sulfate. After evaporation of the solvent, the crude product was
purified by thin layer chromatography on silica gel (hexane/EtOAc = 2/1) to afford 2 as a white solid (17 mg,
77%).
[α]D23 16.8(c 0.89, CHCl3)
1
IR (ATR) 3382, 3293, 3057, 3031, 2929, 2858, 1732, 1683 cm
mp 75–77 ºC.
1H NMR (500 MHz, CDCl3) δ 7.46–7.13 (m, 30H, Trt), 6.56 (d, J = 7.5 Hz, 1H, NH), 5.54 (d, J = 7.5 Hz,
12
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10.1002/ejoc.202000459
European Journal of Organic Chemistry
1H, NH), 4.99–4.92 (m, 1H, 16-H), 4.36 (td, J = 7.5, 5.5 Hz, 1H, 6-H), 4.02 (q, J = 5.5 Hz, 1H, 3-H), 2.86
(dd, J = 12.5, 7.5 Hz, 1H, 1'''-H), 2.71 (dd, J = 12.0, 5.5 Hz, 1H, 1''-H), 2.66 (dd, J = 12.0, 5.5 Hz, 1H,
1''-H), 2.53 (dd, J = 12.5, 5.5 Hz, 1H, 1''-H), 2.22 (ddd, J = 14.5, 5.5, 3.5 Hz, 1H, 9-H), 1.91–1.83 (m, 1H,
9-H), 1.72–0.95 (m, 16 H, 10-H , 11-H, 12-H, 13-H, 14-H, 15-H, 1'-H, 2'-H), 0.87 (t, J = 7.0 Hz, 3H, 3'-H)
13C NMR (125 MHz, CDCl3)173.1, 169.5, 144.4, 144.3, 129.5, 129.4, 128.1, 127.9, 126.9, 126.7, 75.7,
67.4, 66.3, 52.2, 52.1, 36.7, 36.1, 33.4, 33.3, 33.0, 27.2, 26.4, 26.4, 24.8, 22.6, 18.5, 13.9
HRMS (ESI/TOF) m/z [M+Na]+ cacld for C56H60N2O4S2Na 911.3887, found 911.3881.
(9R,14R,17R)-FE399 (1)
A solution of I2 (25 mg, 0.020 mmol) in dichloromethane (32 mL) and methanol (3.6 mL) was added
dropwise to a solution of 2 (18 mg, 0.020 mmol) in dichloromethane (4.0 mL) and methanol (0.4 mL). The
solution was stirred at room temperature for 20 min. The reaction was quenched with saturated aqueous
sodium thiosulfate (11 mL). The organic layer was separated and the aqueous layer was extracted with a
mixed organic solvent (chloroform/methanol = 9/1). The combined organic layer was washed with brine,
and dried over sodium sulfate. After evaporation of the solvent, the crude product was purified twice by thin
layer chromatography on silica gel (chloroform/methanol = 9/1, and 93/7) to afford (9R,14R,17R)-1 as a
1
conformational mixture (5.4 mg, 67%). H NMR of the product showed that the mixture contained
approximately 1.0:0.65 mixture of the two conformers.
mp 263–265ºC (decomposition)
[α]D23 76.2 (c 0.52, CHCl3/MeOH = 9/1)
1
IR (ATR) 3308, 2928, 2856, 1719, 1668, 1654, 1640, 1508 cm
1H NMR (500 MHz, DMSO-d6) major conformer, δ 0.84 (t, J = 7.3 Hz, 3H, 12-H), 0.92-1.60 (m, 16H, 3-H,
4-H, 5-H, 6-H, 7-H, 8-H, 10-H,11-H), 2.11-2.04 (m, 1H, 2-Ha), 2.25-2.16 (m, 1H, 2-Hb), 3.22–3.15 (m, 2H,
15-Ha, 18-Ha), 3.40 (dd, J = 14.8, 10.5 Hz, 15-Hb, 1H), 3.45 (dd, J = 15.0, 3.5 Hz, 18-Hb, 1H), 4.17–4.13
(m, 1H, 17-H), 4.88–4.79 (m, 2H, 9-H, 14-H), 7.74 (d, J = 11.0 Hz, 1H, 14-NH), 8.10 (d, J = 5.5 Hz, 1H,
17-NH); minor conformer, δ 0.84 (t, J = 7.3 Hz, 3H, 12-H), 0.92-1.60 (m, 16H, 3-H, 4-H, 5-H, 6-H, 7-H,
8-H, 10-H,11-H), 1.94 (dt, J = 13.5, 5.2 Hz, 1H, 2-Ha), 2.25-2.16 (m, 1H, 2-Hb), 3.06 (brd, J = 15.0 Hz, 1H,
15-Ha), 3.25 (brd, J = 14.5 Hz, 1H, 18-Ha), 3.63-3.55 (m, 2H, 15-Hb, 18-Hb), 3.95–3.89 (m, 1H, 17-H),
4.88–4.79 (m, 2H, 9-H, 14-H), 8.05 (d, J = 9.0 Hz, 1H, 14-NH), 8.81 (br, 1H, 17-NH);
13C NMR (125 MHz, DMSO-d6)174.8, 173.1, 170.3, 74.1, 53.5, 52.6, 49.3 (minor), 47.4, 44.1, 36.0, 35.6
(minor), 35.3 (minor), 34.9, 33.4 (minor), 32.3, 29.0 (minor), 27.8 (minor), 27.7 (minor), 26.9, 26.62, 26.58,
26.2 (minor), 25.2 (minor), 24.8, 22.6 (minor), 21.6, 18.4 (minor), 18.3, 13.8 (minor), 13.7.
13
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10.1002/ejoc.202000459
European Journal of Organic Chemistry
HRMS (ESI/TOF) m/z [M+Na]+ cacld for C18H30N2O4S2Na 425.1539, found 425.1537
(3R,6R,16R)-16-Propyl-3,6-bis(benzylsulfanylmethyl)-1-oxa-4,7-diazacyclohexadecane-2,5,8-trione
(17)
To a solution of (9R,14R,17R)-1 (1.8 mg, 4.5 μmol) in DMF (0.23 mL) was added TCEP·HCl (1.3 mg, 4.5
μmol) and K2CO3 (3.1 mg, 2.3 μmol). After stirring at room temperature for 1 h, benzyl bromide (1.3 μL,
10.8 μmol) was added. The solution was stirred at room temperature for 12 h. After evaporation of the
solvent, the crude product was purified by thin layer chromatography on silica gel (hexane/EtOAc = 1/1) to
afford 17 as a white solid (3.0 mg, quant.).
[α]D26 33.1(c = 0.20, CHCl3)
1
IR (ATR) 3306, 2929, 2856, 1714, 1656, 1634, 1533 cm
mp 172–173 ºC
1H NMR (500 MHz, CDCl3) δ 735–7.21 (m, 10H, Ph), 6.99 (d, J = 7.0 Hz, 1H, NH), 5.95 (d, J = 7.5 Hz,
1H, NH), 5.07–5.01 (m, 1H, 16-H), 4.68 (dd, J = 13.0, 6.0 Hz, 1H, 6-H), 4.59 (dd, J = 12.5, 5.0 Hz, 1H,
3-H), 3.75–3.66 (m, 4H, Bn), 3.06-2.73 (m, 4H, 1''-H, 1'''-H), 2.33 (ddd, J = 14.0, 6.5, 3.0, Hz, 1H, 9-H),
1.98 (ddd, J = 14.0, 11.5, 3.0 Hz, 1H, 9-H), 1.80–1.06 (m, 16H, 10-H, 11-H, 12-H, 13-H, 14-H, 15-H, 1'-H,
2'-H), 0.89 (t, J = 7.0 Hz, 3H, 3'-H).
13C NMR (125 MHz, CDCl3)173.6, 170.1, 170.0 138.1, 137.9, 129.0, 128.9, 128.8, 128.6, 127.4, 127.2,
75.9, 52.9, 52.1, 37.0, 36.8, 36.6, 36.3, 33.5, 33.1, 32.8, 27.1, 26.5, 26.4, 25.0, 22.5, 18.6, 13.9
HRMS (ESI/TOF) m/z [M+Na]+ cacld for C32H44N2O4S2Na 607.2635, found 607.2664
This study was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education,
Science, Sports and Culture, Japan.
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10.1002/ejoc.202000459
European Journal of Organic Chemistry
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11. Our results were also quite in line with the conclusions reported by Katsuta et al.6
15
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