Total synthesis of (+)-negamycin and its 5-epi-derivative

Article abstract of DOI:10.1016/j.tet.2009.10.097

(+)-Negamycin was prepared in 13 steps in an overall yield of 31% from commercially available ethyl (R)-(+)-4-chloro-3-hydroxybutanoate. The key step in the reaction sequence was a highly stereoselective asymmetric Michael addition of chiral amine (-)-21

Full text of DOI:10.1016/j.tet.2009.10.097

Tetrahedron 66 (2010) 314–320
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Total synthesis of (þ)-negamycin and its 5-epi-derivative
Shigenobu Nishiguchi a, Magne O. Sydnes a, Akihiro Taguchi c, Thomas Regnier a, Tetsuya Kajimoto b,
,c,
Manabu Node b, Yuri Yamazaki c, Fumika Yakushiji c, Yoshiaki Kiso a, Yoshio Hayashi a
*
a Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan
b Pharmaceutical Manufacturing Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan
c Department of Medicinal Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
(þ)-Negamycin was prepared in 13 steps in an overall yield of 31% from commercially available ethyl (R)-
(þ)-4-chloro-3-hydroxybutanoate. The key step in the reaction sequence was a highly stereoselective
asymmetric Michael addition of chiral amine (ꢀ)-21 [(1S,2R)-(ꢀ)-2-methoxybornyl-10-benzylamine]
Received 15 September 2009
Received in revised form 24 October 2009
Accepted 27 October 2009
Available online 31 October 2009
into the
a,b-unsaturated carbonyl moiety of key intermediate 8, thus establishing the second chiral
center in (þ)-negamycin. 5-epi-Negamycin was also prepared in a similar fashion.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
The unusual antibiotic (þ)-negamycin (1) containing a hydra-
zine peptide bond was first isolated by Umezawa et al. in 1970 from
culture filtrates of three strains closely related to Streptomyces
purpeofuscus.1,2 (þ)-Negamycin was found to exhibit inhibitory
activity toward multi drug-resistant Gram-positive and Gram-
negative bacteria,1 and it was also found to be a specific inhibitor of
protein biosynthesis with miscoding activity.3 In 2003 Matsuda
et al. reported that (þ)-negamycin was found to restore dystrophin
expression in skeletal and cardiac muscles of mdx mice, an animal
model of Duchenne muscular dystrophy (DMD).4 The first total
synthesis of (þ)-negamycin was reported in 1972 and this work
confirmed the assigned structure for the natural product.5 Since
this early work, a number of total syntheses have been reported of
racemic negamycin6 and (þ)-negamycin.7 There are also several
reports of formal total synthesis of natural product 1.8 Our own
interest regarding this compound was triggered by the latest
findings by Matsuda and co-workers and an efficient total synthesis
of (þ)-negamycin was recently communicated (Fig 1).9 Herein we
report on our second approach toward this intriguing natural
product starting from commercially available ethyl (R)-(þ)-4-
chloro-3-hydroxybutanoate 6. We also report our synthesis of 5-
epi-negamycin 25, which only has been synthesized by one group
previously.7i
Figure 1. Chemical structure of (þ)-negamycin.
2. Results and discussion
In order to secure an expedient synthesis of (þ)-negamycin 1,
two synthetic routes outlined in the retrosynthetic analysis were
embarked upon (Scheme 1). The analysis suggested that natural
product 1 could be prepared from hydrazine 27i and amino acid
derivative 3 or 7 in route A and B, respectively. Compound 3 in route
A could in its place come from substrate 4 via an new asymmetric
Michael addition reaction10b followed by hydrolysis of the ester
functionality. This stereoselective amination method was recently
developed by Node and co-workers11,12 using chiral methoxy-
bornyl-10-benzylamine as a chiral amine source, and the bulky
tert-butyl ester was a crucial functional moiety to induce high
stereoselectivity. Key intermediate 4 could be derived from alde-
hyde 5, which could be prepared from relatively cheap commer-
cially available chiral ester
6 via several functional group
interconversions. The azide functionality within substrates 5, 4 and
3 was envisioned functioning as a ‘protected’ amine group, which
could be unmasked at the very last step of the synthesis, i.e., the
final deprotection step. Realizing that the azide group could func-
tion as a masked amine meant that the synthetic pathway toward
(þ)-negamycin could be reduced by potentially two steps (pro-
tection of the amino functionality and deprotection). If successful,
* Corresponding author. Tel./fax: þ81 42 676 3275.
E-mail address: yhayashi@toyaku.ac.jp (Y. Hayashi).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.10.097

S. Nishiguchi et al. / Tetrahedron 66 (2010) 314–320
315
The synthesis of (þ)-negamycin 1 started with the conversion of
the commercially available ethyl (R)-(þ)-4-chloro-3-hydroxy-
butanoate 6 over three steps to aldehyde 12 as depicted in Scheme 2,
by using a method developed by Hirai et al. for the synthesis of the
opposite enantiomer of aldehyde 12.13 The chloride functionality
within ester 6 was first converted to the corresponding azide 10
upon treatment with sodium azide in DMF at 100 ꢁC. Attempts to
convert the unprotected alcohol 10 directly to the corresponding
aldehyde only resulted in formation of trace amounts of the desired
product together with a small amount of the corresponding diol. The
free hydroxyl functionality was therefore first protected as the
TBDMS ether 11, which was next treated with DIBAL-H in CH2Cl2 at
ꢀ78 ꢁC, thus forming the desired aldehyde 12.
Route A
OH
NHR3
N
CO2R3
CO2H
(+)-1
+
H2N
3
N3
2
1) amide coupling
2) deprotection
1) Michael amination
2) hydrolysis
OH
OR1
OR1
CO2Et
CHO
CO2tBu
Cl
N3
N3
6
5
4
Due to the slightly unstable nature of aldehyde 12, it was used
directly in the Wittig reaction without purification. Namely, alde-
hyde 12 was treated with (tert-butoxycarbonylmethylene)-
triphenylphosphorane in THF for 1 h to afford the desired ester 13
in 73% yield over 2 steps after purification by flash chromatography
on silica. Proton NMR analysis of substrate 13 revealed that the sole
product of this reaction was the compound with E-geometry as
evident from the large coupling constants for the vinylic protons
(16 Hz). However, the asymmetric Michael addition to compound
14 proceeded in low yield (32%). This might be due to the instability
of substrate 13. From TLC analysis of the crude product 13, it was
clear that this compound was the only product formed during the
Wittig reaction from aldehyde 12. However, compound 13 was
sensitive to the slightly acidic silica gel, which resulted in partly
decomposition during flash chromatography. Ester 13 was also
sensitive toward storage and decomposed to a complex mixture of
compounds rapidly. The unstable nature of ester 13 hampered the
progress of the synthesis via Route A. Hence, the alternative Route B
was considered to be more suitable.
1) substitution
Wittig reaction
2) protection
3) reduction
Route B
O
NHR3
N
CO2R3
N
(+)-1
+
CO2H
H2N
Boc
2
7
1) amide coupling
2) deprotection
1) Michael amination
2) hydrolysis, etc
O
OH
O
N
N
CO2Et
CHO
CO2tBu
Boc
Boc
Cl
6
9
8
1) substitution
2) protection
3) reduction
Wittig reaction
For the strategy outlined in Route B, the use of (R)-(þ)-4-amino-
3-hydroxybutyric acid 16 [nicknamed (R)-(ꢀ)-GABOB], a readily
prepared intermediate, was viewed as a good starting point
(Scheme 3). (R)-(ꢀ)-GABOB has previously been prepared via
a number of routes using various starting materials.14 To this end,
ester 10 was hydrolyzed followed by reduction of the azide func-
Scheme 1. Retrosynthetic analysis of (þ)-negamycin 1.
the strategy outlined below with its eight steps would facilitate
a short total synthesis of (þ)-negamycin. In contrast, in route B the
terminal amino functionality was planned to be constructed in the
early stage of the synthesis, while we envisaged installing the
amino functionality by using a similar asymmetric Michael addition
reaction on the key intermediate olefin 8. Route B required more
steps, including protection and deprotection of the terminal amino
group. However, the unstable and potentially explosive nature of
the azide group in Route A can mostly be avoided.
tionality within compound 15 to (R)-(ꢀ)-GABOB 16 using a litera-
14c
ˆ
ture procedure reported by Larcheveque and Henrot. However,
the yield for the reduction was relatively low (45%) and a different
order of events was found to be more efficient. Reduction of azide
10 under an atmosphere of hydrogen over Pd/C (10%) in the
presence of Boc2O gave the corresponding Boc protected (R)-
(ꢀ)-GABOB derivative 17 in 68% yield. Hydrolysis of the ester group
upon treatment with KOH in ethanol/water 2:1 gave the corre-
sponding acid 18 in an excellent yield (99%). Acid 18 was then
converted to Weinreb amide 19 followed by protection of the free
hydroxyl group and amide nitrogen as an oxazolidinone, thus
forming amide 20 in 95% yield. Weinreb amide 20 was then
converted to aldehyde 9, which was treated directly with (tert-
butoxycarbonylmethylene)triphenylphosphorane in THF under
reflux overnight. This gave, after purification by flash chromatog-
raphy on silica, key intermediate 8 in 80% yield over the two steps.
With compound 8 in hand we were nicely set-up for the asym-
metric Michael addition,10 which we envisioned being executed by
utilizing the methodology developed by Node and co-workers
Scheme 4.11
As reported previously by us, chiral auxiliary (ꢀ)-219,12 was first
converted to the corresponding lithium salt at ꢀ50 ꢁC. Slow addi-
tion of intermediate 8 to the resulting reaction mixture resulted in
smooth conversion to the desired product 22, which was isolated in
96% yield based on recovered starting material (17% of intermediate
8 was recovered from the reaction mixture and recycled) (80% yield
in Ref. 9). Key intermediate 22 was formed as a single diastereomer
(de >99%) as judged by 1H NMR analysis. Interestingly the
Scheme 2. Synthesis of Michael adduct 14 in Route A.

316
S. Nishiguchi et al. / Tetrahedron 66 (2010) 314–320
H2, Pd/C, (Boc)2O,
AcOEt, rt, 12 h
OH
H
N
i) 4 M HCl / dioxane
10
CO2Et
OMe
Boc
67%
17
NBn
ii) (Boc)2O, Et3N
THF
O
KOH,
EtOH/H2O (2:1),
rt, 15 h
N
CO2tBu
KOH,
Boc
EtOH/H2O (2:1),
rt, 2 h
97%
22
OH
99%
N3
CO2H
OH
H
N
CO2H
15
Boc
OMe
18
NBn
O
H2, Pd/C,
OMe
MeOH/H2O (9:1),
rt, 4 h
HCl.HN(OMe)Me,
EDC.HCl, Et3N,
NBn
OH
H
N
45%
DMF, 0 ºC - rt, 15 h
COOH
NHBoc
Boc
O
OH
92%
O
H2N
CO2H
OH
23
24
H
N
OMe
N
16
Boc
19
Scheme 5. Formation of stable d-lactone 24.
DMP, BF3·Et2O,
acetone, reflux,
overnight
convert lactone 24 to the desired product 23 failed. The alternate
procedure, namely removing the chiral auxiliary upon treatment
with NIS in dichloromethane,12 was successful and resulted in the
formation of (þ)-negamycin 1 over five steps using chemistry
recently disclosed by us (Scheme 6).9
DIBAL-H,
95%
toluene,
-78 ºC, 3.5 h
O
O
O
N
N
CHO
OMe
N
Boc
Boc
9
20
Ph3P=CHCO2tBu,
THF, reflux, overnight
80% (2 steps)
CO2tBu
O
N
Boc
8
Scheme 3. Synthesis of key intermediate 8 in Route B.
stereochemistry at the C-5 position did not influence the stereo-
selectivity in our asymmetric Michael addition. The total yield of
Michael adduct 22 from starting material 6 was 41%.
Scheme 6. Synthesis of (þ)-negamycin 1.
With the two chiral centers embodied in (þ)-negamycin (1)
securely in place, focus now shifted toward the latter half of the
synthesis. The question was whether we should hydrolyze the ester
functionality first in order to install the hydrazine moiety prior to
cleave the chiral auxiliary or inversely. To examine these possibil-
ities, compound 22 was treated with 4 M HCl in dioxane, resulting
in hydrolysis of the t-butyl ester functionality as well as removal of
the oxazolidinone and Boc protection groups (Scheme 5). The Boc
group was then reinstalled by treating the crude product mixture
with Boc2O and Et3N in THF. This gave acid 23 in a low yield (25%)
together with the undesired lactone 24 (75% yield). Attempts to
(þ)-Negamycin 1 was by these means prepared in an overall
yield of 31% over 13 steps. The spectroscopic and physical data
obtained for the target compound was in full accord with the data
reported in the literature. Although the overall yield is lower than
our most efficient route to this compound, which uses an expensive
Grubbs’ Catalyst (Second Generation) for the key cross-metathesis
reaction.9 The yield obtained for compound 1 compares favorably
with most previous synthesis of (þ)-negamycin.5,7
For the purpose of preparing a sample of 5-epi-negamycin 25 for
biological testing, the opposite stereoisomer at the 5 position viz.
compound 3R,5S-22 was synthesized according to the previously
reported method.9 Namely, the synthesis was started from achiral
N-Boc-2-aminoacetaldehyde, followed by an asymmetric allylbo-
ration using (ꢀ)-B-allyldiisopinocampheylborane [(ꢀ)-Ipc2B(allyl)]
with the opposite stereochemistry, cross-metathesis reaction
using Grubbs second-generation [Ru-II] catalyst and asymmetric
Michael addition (data not shown). The obtained compound
3R,5S-22 was subjected to the same sequence of reactions as
just described for (þ)-negamycin in order to give 5-epi-neg-
amycin 25 in 41% yield as the TFA salt from substrate 3R,5S-22
(Scheme 7).
Scheme 4. Asymmetric Michael addition using chiral methoxybornyl-10-benzylamine
as a chiral amine source.

S. Nishiguchi et al. / Tetrahedron 66 (2010) 314–320
317
1) NIS, CH2Cl2,
1) KOH, MeOH /
H2O, MW,
100%
66%
OMe
3 steps (46%)
See ref. 9
2) (Boc)2O, THF,
99%
Boc
Boc
NBn
O
NH
O
N
H
CHO
2)
N
CO2tBu
N
CO2tBu
Boc
Boc
O
N
CO2tBu
H2N
26
3R,5S-22
27
EDC. HCl,
HOBt, Et3N,
CH2Cl2, 90%
Boc
4 M HCl /
OH
NH2
dioxane, 73%
NH
O
O
H2N
N
CO2H
N
N
CO2tBu
N
HPLC purification
Lyophilization
Boc
N
H
H
28
5-epi-Negamycin 25
Scheme 7. Synthesis of 5-epi-negamycin.
3. Conclusion
d
172.0, 67.3, 61.0, 55.5, 38.4, 14.1; HRMS (CIþ) calcd for C6H12N3O3
(MþþH) 174.0878, found 174.0875.
The synthetic strategy outlined here resulted in the preparation
of (þ)-negamycin in 13 steps with an overall yield of 31% starting
from commercially available chiral starting material. By utilizing
the same synthetic strategy a sample of 5-epi-negamycin was
prepared. This synthetic strategy together with our previously
reported approach9 is able to facilitate the preparation of a range of
analogues of the natural product for biological evaluation. Work
toward this end is currently ongoing in our laboratories.
4.1.2. Ethyl (3R)-4-azido-3-tert-butyldimethylsilyloxybutanoate 11.
Imidazole (47.9 mg, 0.70 mmol) was added to a stirred solution of
alcohol 10 (52.5 mg, 0.303 mmol) and tert-butyldimethylsilyl
chloride (88.1 mg, 0.584 mmol) in DMF (2.5 mL) at room tem-
perature. The resulting reaction mixture was then stirred at room
temperature for 18 h before being diluted with EtOAc (15 mL) and
washed with water/brine (5ꢂ10 mL of a 1:1 solution) and brine
(1ꢂ10 mL) before being dried (MgSO4). Filtration and concentration
under reduced pressure gave a light-yellow oil, which was sub-
jected to flash column chromatography (silica, n-hexane/n-
hexane/EtOAc¼9:1 gradient eluent). Concentration of the rele-
4. Experimental
4.1. General procedures
vant fractions (Rf 0.4 in n-hexane/EtOAc¼9:1) gave the title
25
compound 11 (86.9 mg, quant.) as a clear colorless oil. [
a
]
þ2.2ꢁ
Melting points were measured on a Yanagimoto micro hot-stage
apparatus and are uncorrected. Proton (1H) and carbon (13C) NMR
spectra were recorded on either a JEOL JNM-AL300 spectrometer
operating at 300 MHz for proton and 75 MHz for carbon, or a Varian
UNITY INOVA 400NB spectrometer operating at 400 MHz for pro-
ton and 100 MHz for carbon. Chemical shifts were recorded as
D
(c 0.33, CHCl3); 1H NMR (400 MHz, CDCl3)
d
4.23 (dt, J¼6.2 and
5.2 Hz, 1H), 4.13 (dd, J¼2.4 and 7.1 Hz, 2H), 3.37 (dd, J¼4.3 and
13 Hz, 1H), 3.24 (dd, J¼5.3 and 13 Hz, 1H), 2.54 (dd, J¼3.8, and
6.2 Hz, 2H), 1.27 (t, J¼7.1 Hz, 3H), 0.89 (s, 9H), 0.13 (s, 3H), 0.09 (s,
3H); 13C NMR (100 MHz, CDCl3)
d 170.8, 68.7, 60.6, 56.4, 40.1,
25.6, 17.9, 14.1,ꢀ4.2, ꢀ5.7; MS (CIþ) m/z 288 (MþþH, 14%), 272
(12), 245 (32), 231 (48), 230 (100), 156 (8), 115 (22), 73 (60);
HRMS (CIþ) calcd for C12H26N3O3Si (MþþH) 288.1743, found
288.1739.
d
values in parts per million (ppm) downfield from tetramethylsi-
lane (TMS). Low- and high-resolution mass spectra (CI and EI) were
recorded on a JEOL JMS-GCmate. Optical rotations were measured
with a Horiba High-speed Accurate Polarimeter SEPA-300 at the
sodium-D line (589 nm) at the concentrations (c, g 100 mLꢀ1). The
measurements were carried out between 24–28 ꢁC in a cell with
4.1.3. tert-Butyl (5R)-6-azido-5-tert-butyldimethylsilyloxy-(2E)-hex-
enoate 13. DIBAL-H (293 mL of a 1.02 M solution in hexane,
path length (l) of 0.5 dm. Specific rotations [
a
]
are given in
D
10ꢀ1 deg cm2 gꢀ1. Preparative HPLC was performed using a C18
reverse-phase column (25,020 mm; YMC-Pack ODS-AM) with the
binary solvent system.
0.299 mmol) was added dropwise to a solution of ester 11
(66.0 mg, 0.230 mmol) in dry CH2Cl2 (7.7 mL) at ꢀ78 ꢁC. The re-
action mixture was stirred at ꢀ78 ꢁC for 10 min, before the reaction
mixture was quenched with water (10 mL) and stirred vigorously
for 5 min. The mixture solution was filtered through a pad of
CeliteÒ with CHCl3 and washed with H2O, brine. The combined
organic layer was dried over Na2SO4. Filtration and concentration
under reduced pressure gave as an unclear oil. The product was
used directly in the next reaction without further purification.
4.1.1. Ethyl (R)-4-azido-3-hydroxybutanoate 10. Sodium azide
(2.77 g, 42.6 mmol) was added to a stirred solution of ethyl (R)-
(þ)-4-chloro-3-hydroxybutanoate 6 (3.50 g, 21.0 mmol) in DMF
(55 mL) at room temperature. The resulting reaction mixture was
then heated at 100 ꢁC for 3 h before being cooled to room tem-
perature and filtered. The remaining salt was washed with ether
(3ꢂ10 mL) and filtered. The combined organic fractions were con-
centrated under reduced pressure and the resulting yellow oil was
subjected to flash column chromatography (silica, n-hexane/
EtOAc¼8:2/7:3 gradient eluent). Concentration of the relevant
fractions (Rf 0.3 in n-hexane/EtOAc¼7:3) under reduced pressure
gave the title azide 1013 (3.28 g, 90%) as a light-yellow oil.
(tert-Butoxycarbonylmethylene)triphenylphosphorane
(113 mg,
0.299 mmol) was added to a stirred solution of aldehyde 12 in THF
(2.3 mL) at room temperature. The reaction mixture was then
stirred at room temperature for 1 h. The solvent was removed
under reduced pressure and extracted with EtOAc (15 mL) and
washed with satd NaHCO3 aq (2ꢂ10 mL), brine (1ꢂ10 mL) before
being dried over Na2SO4. Filtration and concentration under re-
duced pressure gave a yellow oil, which was subjected to flash
column chromatography (silica, n-hexane/EtOAc¼9:1). Concen-
tration of the relevant fractions (Rf 0.8 in n-hexane/EtOAc¼2:1)
gave the title compound 13 (57.2 mg, 73% over 2 steps) as
28
20
[
a]
þ7.5ꢁ (c 3.65, MeOH), lit.13
[a
]
þ7.4ꢁ (c 4.05, MeOH); 1H NMR
D
D
(300 MHz, CDCl3)
d
4.19 (q, J¼7.2 Hz, 2H), 4.12 (partly obscured m,
1H), 3.35 (ddd, J¼12, 6.0 and 4.6 Hz, 2H), 3.31 (s,1H), 2.54 (dd, J¼2.9
and 0.7 Hz, 2H), 1.29 (t, J¼7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3)

318
S. Nishiguchi et al. / Tetrahedron 66 (2010) 314–320
26
a colorless oil. [
a
]
ꢀ21.7ꢁ (c 1.00, CHCl3); 1H NMR (300 MHz,
(100 mg) were added to a stirred solution of alcohol 10 (1.00 g,
5.78 mmol) in EtOAc (60 mL) at room temperature. The resulting
reaction mixture was subjected to three cycles of vacuum followed by
flush with H2 before being stirred vigorously under an atmosphere of
H2 for 12 h. The reaction mixture was then filtered through a pad of
CeliteÒ and washed with EtOAc (3ꢂ10 mL). The resulting filtrate was
concentrated under reduced pressure and the resulting residue was
subjected to flash column chromatography (silica, n-hexane/
D
CDCl3)
d
6.77 (dt, J¼7.7 and 16 Hz, 1H), 5.79 (dt, J¼1.4 and 16 Hz,
1H), 3.89 (dt, J¼10 and 5.4 Hz, 1H), 3.27 (dd, J¼4.4 and 13 Hz, 1H),
3.16 (dd, J¼5.8 and 13 Hz, 1H), 2.43–2.36 (m, 2H), 1.48 (s, 9H), 0.91
(s, 9H), 0.12 (s, 3H), 0.09 (s, 3H); HRMS (ESþ) calcd for
C16H32N3O3Si (MþþH) 342.2213, found 342.2207.
4.1.4. tert-Butyl (5R,3R)-6-azido-5-tert-butyldimethylsilyloxy-3-(N-
benzyl-N-(2-methoxy-7,7-dimethylbicyclo[2.2.1]heptan-1-ylmethyl)-
amino)hexanoate 14. A 1.55 M solution of n-butyllithium in hexane
EtOAc¼2:1). Concentration of the relevant fractions (Rf 0.3) gave the
25
desired compound 17 (0.95 g, 67%) as a light-yellow oil. [
a
]
þ6.6ꢁ (c
D
20
(290
m
L) was added dropwise to a solution of (ꢀ)-21 (122 mg,
1.06, CHCl3); lit.7j
CDCl3)
[
a
]
þ6.6ꢁ (c 1.08, CHCl3); 1H NMR (400 MHz,
D
0.448 mmol) in dry THF (1.5 mL) and the mixture was stirred at
ꢀ50 ꢁC. After stirring for 30 min, a solution of 13 (76.6 mg,
0.224 mmol) in anhydrous THF (1.2 mL) was added dropwise to the
reaction mixture, which was stirred at ꢀ40 ꢁC for another 1 h. The
reaction was quenched by adding a satd NaHCO3 aq and the mixture
was extracted with ether. The organic layer was washed with brine
and dried over Na2SO4. After the solvent was concentrated under
reduced pressure. The residue was purified by flash column
chromatography (n-hexane/EtOAc¼12:1) to afford desired product
14 (43.8 mg, 32%) as a colorless oil. 1H NMR (300 MHz, CDCl3)
d
4.96 (br s, 1H), 4.18 (q, J¼7.1 Hz, 2H), 4.14–4.06 (m, 1H), 3.46
(br s, 1H), 3.38–3.28 (m, 1H), 3.12 (ddd, J¼5.7, 6.8 and 12 Hz, 1H), 2.49
(dd, J¼2.4 and 5.6 Hz, 2H), 1.48 (s, 9H), 1.28 (t, J¼7.1 Hz, 3H); 13C NMR
(100 MHz, CDCl3) d 172.6, 156.5, 79.6, 67.7, 60.9, 45.5, 38.6, 28.3, 14.1;
HRMS (CIþ) calcd for C11H22NO5 (MþþH) 248.1498, found 248.1504.
4.1.8. (R)-4-(tert-Butyloxycarbonyl)amino-3-hydroxybutanoic acid
18. KOH (1.36 g, 24.2 mmol) was added to a stirred solution of al-
cohol 17 (2.00 g, 8.00 mmol) in EtOH (24.5 mL) and water (12.3 mL)
at room temperature. The resulting reaction mixture was stirred at
room temperature for 2 h before EtOH was removed under reduced
pressure. The resulting aqueous phase was acidified to pH 1 by
addition of 10% citric acid aq and extracted with EtOAc (3ꢂ20 mL).
The combined organic fraction was washed with brine (2ꢂ20 mL)
before being dried (Na2SO4). The crude residue obtained after fil-
tration and concentration under reduced pressure was subjected to
flash column chromatography (silica, CHCl3/MeOH¼10:1). Con-
d
7.39–7.28 (m, 4H), 7.25–7.18 (m, 1H), 3.85 (d, J¼14 Hz, 1H), 3.77–
3.69 (m, 1H), 3.39–3.34 (m, 1H), 3.33–3.30 (m, 1H), 3.28 (t, J¼3.0 Hz,
1H), 3.13 (s, 3H), 3.18–2.92 (m, 2H, including 1H at
and 6.2 Hz), 2.72 (d, J¼14 Hz,1H), 2.70 (dd, J¼15 and 3.2 Hz,1H), 2.21
(d, J¼14 Hz, 1H), 2.05 (dd, J¼15 and 9.5 Hz, 1H), 1.92–1.72 (m, 2H),
1.69–1.48 (m, 5H), 1.43 (s, 9H), 1.32–1.21 (m, 1H), 1.06–0.97 (m, 1H),
0.92 (s, 3H), 0.85 (s, 9H), 0.76 (s, 3H), 0.04, (s, 3H), ꢀ0.06 (s, 3H);
HRMS (ESþ) calcd for C34H59N4O4Si (MþþH) 615.4306, found
615.4320.
d
3.05, dd, J¼13
centration of the relevant fractions (Rf 0.4 in CHCl3/MeOH¼5:1)
26
gave acid 18 (1.78 g, 99%) as a white solid. Mp 94–95 ꢁC; [
a]
þ9.7ꢁ
D
(c 1.09, CHCl3); 1H NMR (400 MHz, CDCl3)
d 6.12 (br s, 1H), 5.18 (br s,
4.1.5. (R)-4-Azido-3-hydroxybutanoic acid 1514c. Potassium hy-
droxide (1.72 g, 30.7 mmol) was added to a stirred solution of azide
10 (1.77 g, 10.2 mmol) in water (15 mL) and ethanol (30 mL) at
room temperature. The reaction mixture was then stirred at room
temperature for 15 h before ethanol was evaporated under reduced
pressure. The resulting residue was extracted with ether (1ꢂ10 mL)
and the water phase was acidified to pH 1 by addition of 3 M HCl.
Water was then removed under reduced pressure and the residue
thus obtained was dissolved in EtOAc and dried over MgSO4. Fil-
tration and concentration in vacuo gave the title acid 1514c (1.44 g,
1H), 4.16–4.11 (m, 1H), 3.39–3.28 (m, 1H), 3.16 (ddd, J¼1.3, 6.0 and
6.6 Hz, 1H), 2.53 (dd, J¼5.3 and 2.2 Hz, 2H), 1.45 (s, 9H); 13C NMR
(100 MHz, CDCl3)
d 175.8, 157.0, 80.2, 67.8, 45.1, 38.5, 28.3; HRMS
(CIþ) calcd for C9H18NO5 (MþþH) 220.1185, found 220.1184.
4.1.9. (R)-4-(tert-Butyloxycarbonyl)amino-3-hydroxy-N-methoxy-N-
methylbutanamide 19. Triethylamine (2.00 mL, 14.6 mmol) was
added dropwise to a solution of acid 18 (2.19 g,13.3 mmol) and N,O-
dimethylhydroxylamine hydrochloride (1.42 g, 14.6 mmol) in DMF
(70 mL) maintained at 0 ꢁC. The resulting reaction mixture was
then stirred for 0.1 h at 0 ꢁC before EDC$HCl (3.06 g,16.0 mmol) was
added. The reaction mixture was then allowed to heat to room
temperature and stirred for 15 h before the solvent was removed
under reduced pressure. The resulting residue was diluted with
EtOAc (70 mL) and washed consecutively with 10% citric acid aq
(1ꢂ20 mL), satd NaHCO3 aq (1ꢂ20 mL), and brine (1ꢂ20 mL) before
being dried over Na2SO4. Filtration and concentration under re-
duced pressure gave a light-yellow oil, which was purified by flash
column chromatography (silica, CHCl3/MeOH¼10:1). Evaporation
25
20
97%) as a yellow oil. [
a
]
þ2.7ꢁ (c 6.78, MeOH), lit.14c
[
a
]
þ3.02ꢁ (c
D
D
2.55, MeOH); 1H NMR (300 MHz, CDCl3)
d 6.80–5.80 (br s, 2H),
4.27–4.20 (m, 1H), 3.42 (dd, J¼4.4 and 13 Hz, 1H), 3.37 (dd, J¼6.1
and 13 Hz, 1H), 2.63 (d, J¼3.5 Hz, 1H), 2.61 (d, J¼1.1 Hz, 1H); 13C
NMR (100 MHz, CDCl3)
d
176.7, 67.1, 55.5, 38.2; HRMS (ESþ) calcd
for C4H7N3O3Na (MþþNa) 168.0385, found 168.0372.
4.1.6. (R)-4-Amino-3-hydroxybutanoic acid [(ꢀ)-GABOB] 1614c. A
suspension of acid 15 (1.41 g, 9.69 mmol) and Pd/C (10%) (104 mg) in
methanol (29 mL) and water (3.2 mL) was stirred under an atmo-
sphere of hydrogen at room temperature for 4 h. The reaction mix-
ture was then filtered through a pad of CeliteÒ and the filtrate was
concentrated under reduced pressure. The resulting viscous yellow
oil was dissolved in a small amount of water and dilution with ab-
solute alcohol to give the title product as a brown solid. Re-
of the relevant fractions (Rf 0.6 in CHCl3/MeOH¼5:1) gave the de-
27
sired compound 19 (3.21 g, 92%) as a colorless oil. [
a
]
D
þ27.8ꢁ (c
1.20, CHCl3); 1H NMR (400 MHz, CDCl3)
d 5.07 (br s, 1H), 4.16 (br s,
1H), 4.14–4.08 (m, 1H), 3.69 (s, 3H), 3.40–3.32 (m, 1H), 3.19 (s, 3H),
3.18–3.11 (m, 1H), 2.68 (d, J¼17 Hz, 1H), 2.52 (dd, J¼9.2 and 17 Hz,
1H), 1.45 (s, 9H); 13C NMR (100 MHz, CDCl3)
d 173.2, 156.4, 79.4,
crystallization from water/methanol gave the title acid 1614c
67.6, 61.3, 45.5, 35.3, 31.8, 28.3; HRMS (CIþ) calcd for C11H23N2O5
(522 mg, 45%) as awhite solid. Mp 214–215 ꢁC, lit.14c Mp 212 ꢁC; [
a]
(MþþH) 263.1607, found 263.1611.
25
D
20
ꢀ16.1ꢁ (c 0.69, H2O), lit.14c
[
a
]
D
ꢀ20ꢁ (c 2.37, H2O); 1H NMR
(400 MHz, D2O)
d
4.21 (ddt, J¼3.2, 6.6 and 9.4 Hz,1H), 3.17 (dd, J¼3.2
4.1.10. 2-((R)-3-tert-Butyloxycarbonyl-2,2-dimethyloxazolidin-5-yl)-
N-methoxy-N-methylacetamide 20. 2,2-Dimethoxypropane (6.02 mL,
49.1 mmol) and BF3$Et2O (77.1 mL, 0.61 mmol) were added to a stir-
red solution of amide 19 (1.61 g, 6.14 mmol) in acetone (60 mL) at
room temperature. The resulting reaction mixture was then heated
at reflux overnight before being cooled to room temperature and
poured into a solution of satd NaHCO3 aq (30 mL). Acetone was then
and 13 Hz,1H), 2.95 (dd, J¼9.4 and 13 Hz,1H), 2.44 (d, J¼6.6 Hz, 2H);
MS (CIþ) m/z 120 (MþþH, 100%), 103 (18), 102 (46), 84 (66), 57 (62);
HRMS (CIþ) calcd for C4H10NO3 (MþþH) 120.0661, found 120.0657.
4.1.7. Ethyl 4-(N-tert-butyloxycarbonyl)amino-(R)-3-hydroxybutanoate
17. Di-tert-butyl dicarbonate (1.51 g, 6.93 mmol) and Pd/C (10%)

S. Nishiguchi et al. / Tetrahedron 66 (2010) 314–320
319
removed under reduced pressure and the resulting residue was
extracted with EtOAc (3ꢂ30 mL). The combined organic fraction was
washed consecutively with satd NaHCO3 aq (1ꢂ20 mL), and brine
(1ꢂ20 mL) before being dried (Na2SO4). Filtration and concentration
under reduced pressure gave a yellow oil, which was subjected to
flash column chromatography (silica, CHCl3/MeOH¼15:1). Concen-
with 4 M HCl/dioxane (5 mL) at 0 ꢁC. The solution mixture was
stirred at 60 ꢁC for 1.5 h to completely remove three protective
groups, i.e., Boc, dimethylacetal and tert-butyl ester. The solvent
was removed under reduced pressure, and the resultant residue
was used for the next reaction without further purification. The
residue was dissolved in THF (5.7 mL), then Et3N (160 mL,
tration of the relevant fractions (Rf 0.6 in n-hexane/EtOAc¼1:1) gave
1.15 mmol) and (Boc)2O (150 mg, 0.689 mmol) were added to this
solution at 0 ꢁC. The mixture was then stirred at room temperature
for 3.5 h and the solvent was evaporated under reduced pressure.
The resulting residue was extracted with EtOAc and the organic
layer was washed with 10% citric acid aq, satd NaHCO3 aq and brine,
dried over Na2SO4. Filtration and concentration under reduced
pressure gave a crude oil, which was purified by flash column
chromatography (CHCl3/MeOH¼60:1) to give the desired com-
pound 23 (77.3 mg, 25%) as a white solid. An undesired lactone 24
25
desired compound 20 (1.73 g, 95%) as a light-yellow oil. [
a
]
ꢀ18.4ꢁ
D
(c 1.22, CHCl3); 1H NMR (400 MHz, CDCl3)
d 4.49 (br s, 1H), 3.87 (dd,
J¼5.6 and 10 Hz, 1H), 3.70 (s, 3H), 3.19 (s, 3H), 3.14–3.10 (m, 1H),
3.05–2.88 (m, 1H), 2.61 (dd, J¼7.6 and 16 Hz, 1H), 1.58 (s, 3H), 1.51 (s,
3H), 1.46 (s, 9H); 13C NMR (100 MHz, CDCl3)
d 171.0, 152.0, 93.1, 79.7,
70.2, 61.3, 51.2, 36.2, 31.9, 28.4, 26.7, 24.7; HRMS (CIþ) calcd for
C14H27N2O5 (MþþH) 303.1920, found 303.1925.
4.1.11. (E)-tert-Butyl 4-((R)-3-tert-butyloxycarbonyl-2,2-dimethylox-
azolidin-5-yl)but-2-enoate 8. DIBAL-H (15.8 mL of a 0.99 M solution
in toluene, 16.0 mmol) was added dropwise to a stirred solution of
amide 20 (3.72 g, 12.3 mmol) in anhydrous toluene (123 mL)
maintained under and argon atmosphere at ꢀ78 ꢁC. The reaction
mixture was then stirred for 3.5 h at ꢀ78 ꢁC before being quenched
by addition of ice-cold 1 M HCl. The aqueous phase was then
extracted with EtOAc (3ꢂ20 mL) and the combined organic fraction
was washed with satd NaHCO3 aq (1ꢂ30 mL), and brine (1ꢂ30 mL)
before being dried over Na2SO4. Filtration and concentration under
reduced pressure gave 2-((R)-3-tert-butyloxycarbonyl-2,2-dime-
thyloxazolidin-5-yl)acetaldehyde 97j (2.95 g, 12.1 mmol) as a color-
less oil. The product was used directly in the next reaction without
further purification. tert-Buthoxycarbonylmethylenephenyl phos-
phorane (5.02 g, 13.3 mmol) was added to a stirred solution of
aldehyde 9 (2.95 g, 12.1 mmol) in THF (121 mL) at room tempera-
ture. The reaction mixture was then heated at refluxed temperature
for overnight before being cooled to rt. The reaction mixture was
concentrated under reduced pressure and the resulting residue was
diluted with EtOAc (80 mL) and washed consecutively with satd
NaHCO3 aq (1ꢂ30 mL), brine (1ꢂ30 mL) before being dried over
Na2SO4. Filtration and concentration under reduced pressure gave
a light-yellow oil, which was purified by flash column chroma-
tography (silica, n-hexane/EtOAc¼10:1). Concentration of the rel-
was also produced as a colorless oil in a higher yield (215 mg, 75%).
25
Compound 23; [
a
]
ꢀ51.5ꢁ (c 1.00, CHCl3); HRMS (ESþ) calcd for
D
C29H47N2O6 (MþþH) 519.3434, found 519.3397. Compound 24; 1H
HMR (400 MHz, CDCl3) d 7.34–7.28 (m, 4H), 7.25–7.12 (m, 1H), 4.99
(br s, 1H), 4.15 (t, J¼2.4 Hz, 1H), 3.80 (d, J¼15 Hz, 1H), 3.61 (d,
J¼15 Hz, 1H), 3.48 (dd, J¼7.3 and 3.2 Hz, 1H), 3.36 (dd, J¼7.3 and
3.2 Hz, 1H), 3.23–3.10 (m, 5H, including 3H at
d 3.16, s), 2.81 (d,
J¼7.0 Hz, 1H), 2.78 (dd, J¼18 and 9.2 Hz, 1H), 2.62 (dd, J¼18 and
6.2 Hz, 1H), 2.20 (d, J¼14 Hz, 1H), 1.99 (dt, J¼14 and 2.4 Hz, 1H),
1.83–1.75 (m, 1H) 1.67–1.53 (m, 5H), 1.43 (s, 9H), 1.26–1.20 (m, 1H),
1.03–0.92 (m, 4H, including 3H at
(100 MHz, CDCl3) 172.1, 160.0, 140.3, 128.4, 128.0, 126.9, 85.6, 79.7,
d
0.96, s), 0.77 (s, 3H); 13C NMR
d
78.1, 55.8, 55.0, 52.9, 52.7, 47.5, 45.9, 45.3, 44.7, 37.5, 31.4, 31.2, 28.9,
28.4, 27.3, 20.8, 20.4; HRMS (ESþ) calcd for C29H45N2O5 (MþþH)
501.3328, found 501.3346.
4.1.14. (þ)-Negamycin (2-[(3R,5R)-3,6-diamino-5-hydroxyhexanoyl]-
1-methylhydrazinoacetic acid) 1. (þ)-Negamycin was prepared from
compound 22 using our previously reported method.9 The data for
the compound was in full accord with the data reported previously.
4.1.15. (S)-tert-Butyl-5-{(R)-3-(tert-butoxycarbonyl)-2-[N-benzyl-N-
((2-methoxy-7,7-dimethylbicyclo[2,2,1]-heptan-1-yl)methyl)ami-
no]propyl}-2,2-dimethyloxazolidine-3-carboxylate 3R,5S-22. The
25
evant fractions (Rf 0.4 in n-hexane/EtOAc¼5:1) gave the desired
reaction was conducted as outlined in Ref. 9. [
a
]
þ12.9ꢁ (c 1.10,
D
25
compound 8 (1.95 g, 80% over 2 steps) as a colorless oil. [
a
]
D
ꢀ26.2ꢁ
CHCl3); 1H NMR (400 MHz, CDCl3)
d 7.36–7.28 (m, 4H), 7.25–7.19
(c 1.06, CHCl3); 1H NMR (400 MHz, CDCl3)
d
6.82 (ddd, J¼14, 15 and
(m, 1H), 4.19 (br s, 1H), 3.91 (d, J¼20 Hz, 1H), 3.44 (br s, 1H), 3.27
(d, J¼14 Hz, 1H), 3.15 (s, 3H), 2.95–2.78 (m, 4H, including 1H at
16 Hz, 1H), 5.85 (d, J¼16 Hz, 1H), 4.19–4.14 (m, 1H), 3.73–3.65 (m,
1H), 3.13–3.04 (m, 1H), 2.55–2.42 (m, 2H), 1.57 (br s, 3H), 1.54 (br s,
d
2.72, dd, J¼2.7 and 14 Hz), 2.62–2.55 (m, 1H), 2.30 (d, J¼13 Hz,
3H), 1.48 (s, 18H); 13C NMR (100 MHz, CDCl3)
d
165.5, 152.2, 141.9,
1H), 2.05 (dd, J¼10 and 14 Hz, 1H), 1.93 (t, J¼11 Hz, 1H), 1.83 (d,
J¼11 Hz, 1H), 1.73–1.56 (m, 6H), 1.50–1.25 (m, 24H), 1.03 (t,
J¼9.4 Hz, 1H), 0.95 (s, 3H), 0.80 (s, 3H); 13C NMR (100 MHz, CDCl3)
125.8, 93.5, 80.4, 79.6, 72.1, 50.5, 35.6, 28.4, 28.1, 26.7, 24.8; HRMS
(EIþ) calcd for C18H31NO5 (Mþ) 341.2202, found 341.2200.
d
172.2, 152.0, 140.7, 130.0, 128.0, 127.9, 127.0, 92.8, 87.9, 85.6, 80.4,
4.1.12. (R)-tert-Butyl-5-{(R)-3-(tert-butoxycarbonyl)-2-[N-benzyl-N-
((2-methoxy-7,7-dimethylbicyclo[2,2,1]heptan-1-yl)methyl)amino]-
79.0, 77.2, 71.6, 55.9, 54.9, 53.9, 52.9, 50.6, 47.8, 45.4, 45.0, 37.5,
36.5, 34.1, 31.3, 28.5, 28.2, 27.3, 24.2, 20.9, 20.5; HRMS (ESþ) calcd
for C36H59N2O6 (MþþH) 615.4373, found 615.4382.
propyl}-2,2-dimethyloxazolidine-3-carboxylate 22. The reaction was
25
conducted as outlined in Ref. 9. [
(400 MHz, CDCl3)
a]
ꢀ14.0ꢁ (c 1.01, CHCl3); 1H NMR
D
d
7.29–7.26 (m, 4H), 7.22–7.18 (m, 1H), 4.31 (br s,
4.1.16. (S)-tert-Butyl-5-{(R)-3-(tert-butoxycarbonyl)-2-tert-butoxy-
1H), 3.83 (d, J¼14 Hz, 1H), 3.67–3.63, 3.57–3.53 (m, total 1H), 3.47
(dd, J¼7.2 and 2.8 Hz, 1H), 3.32–3.27 (m, 1H), 3.25, 3.21 (s, total 1H),
3.18 (s, 3H), 3.01–2.93 (m, 2H), 2.81–2.70 (m, 2H), 2.18 (d, J¼14 Hz,
1H), 2.12–2.03 (m,1H),1.79–1.73 (m, 2H),1.65–1.26 (m,12H),1.47 (s,
9H), 1.43 (s, 9H), 0.91 (s, 3H), 0.63 (s, 3H); 13C NMR (100 MHz,
carbonylaminopropyl}-2,2-dimethyloxazolidine-3-carboxylate
25
27. The reaction was conducted as outlined in Ref. 9. [
a
]
þ26.7ꢁ
D
(c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3)
d
5.10 (br s, 1H), 4.18–
4.07 (m, 1H), 3.98 (br s, 1H), 3.73 (br s, 1H), 3.04 (t, J¼8.7 Hz, 1H),
2.55–2.42 (m, 2H), 1.91–1.74 (m, 2H), 1.53 (s, 6H), 1.50–1.34 (m,
CDCl3)
d
172.4, 152.4, 140.7, 129.2, 128.0, 126.6, 93.2, 92.8, 85.0, 80.2,
32H); 13C NMR (100 MHz, CDCl3)
d 170.7, 155.3, 152.0, 146.8, 93.3,
79.8, 79.2, 70.7, 70.1, 54.8, 54.0, 52.6, 51.3, 50.9, 47.7, 45.5, 30.8, 28.5,
28.1, 27.4, 27.2, 26.4, 25.3, 24.4, 20.6, 20.4; HRMS (EIþ) calcd for
C36H58N2O6 (Mþ) 614.4294, found 614.4286.
85.2, 81.1, 79.3, 71.5, 51.1, 45.7, 40.7, 38.0, 28.5, 28.4, 27.5, 25.3;
HRMS (ESþ) calcd for C23H47N2O7Na (MþþNa) 481.2914, found
481.2890.
4.1.13. (3R)-3-(Benzyl((2-methoxy-7,7-dimethylbicyclo[2.2.1]heptan-
1-yl)methyl)amino)-6-(tert-butoxycarbonyl amino)-5-hydroxyhex-
anoic acid 23. Compound 22 (353 mg, 0.574 mmol) was treated
4.1.17. (S)-tert-Butyl-(R)-5-[2-(tert-butoxycarbonylamino)-3-{(tert-
butoxycarbonylmethyl)methylaminocarbamoyl}propyl]-2,2-dimethy-
loxazolidine-3-carboxylate 28. The reaction was conducted as

320
S. Nishiguchi et al. / Tetrahedron 66 (2010) 314–320
26
outlined in Ref. 9. [
CDCl3)
a
]
þ15.5ꢁ (c 1.00, CHCl3); 1H NMR (400 MHz,
References and notes
D
d 7.88, 7.36 (br s, total 1H), 5.55–5.30 (m, 1H), 4.16–3.92
1. Hamada, M.; Takeuchi, T.; Kondo, S.; Ikeda, Y.; Naganawa, H.; Maeda, K.; Okami,
Y.; Umezawa, H. J. Antibiot. 1970, 23, 170–171.
2. Kondo, S.; Shibahara, S.; Takahashi, S.; Maeda, K.; Umezawa, H. J. Am. Chem. Soc.
1971, 93, 6305–6306.
3. Uehara, Y.; Kondo, S.; Umezawa, H.; Suzukake, K.; Hori, M. J. Antibiot. 1972, 25,
685–688.
4. Arakawa, M.; Shiozuka, M.; Nakayama, Y.; Hara, T.; Hamada, M.; Kondo, S.;
Ikeda, D.; Takahashi, Y.; Sawa, R.; Nonomura, Y.; Sheykholeslami, K.; Kondo, K.;
Kaga, K.; Kitamura, T.; Suzuki-Miyagoe, Y.; Takeda, S.; Matsuda, R. J. Biochem.
2003, 134, 751–758.
(m, 2H), 3.71 (br s, 1H), 3.60–3.31 (
d, J¼18 Hz, total, 2H), 3.08 (t, J¼9.6 Hz, 1H), 2.98–2.51 (m, 4H
including 3H at
2.74, d, J¼14 Hz), 2.38 (d, J¼5.4 Hz, 1H), 2.04–
1.78 (m, 2H), 1.60 (s, 3H), 1.50–1.32 (m, 30H); 13C NMR (100 MHz,
CDCl3) 174.4, 170.2, 169.6, 155.4, 151.9, 93.7, 82.3, 79.0, 71.5,
58.9, 50.9, 45.7, 43.9, 38.3, 36.9, 28.4, 28.3, 28.1, 26.8, 24.7; HRMS
(ESþ) calcd for C26H48N4O8Na (MþþNa) 567.3370, found
567.3391.
d
3.53, d, J¼20 Hz and
d 3.35,
d
d
5. Shibahara, S.; Kondo, S.; Maeda, K.; Umezawa, H.; Ohno, M. J. Am. Chem. Soc.
1972, 94, 4353–4354.
6. (a) Streicher, W.; Reinshagen, H.; Turnowski, F. J. Antibiot. 1978, 31, 725–728; (b)
Pasquet, G.; Boucherot, D.; Pilgrim, W. R.; Wright, B. Tetrahedron Lett. 1980, 21,
931–934; (c) Pierdet, A.; Ne´de´lec, L.; Delaroff, V.; Allais, A. Tetrahedron 1980, 36,
1763–1772.
7. (a) Wang, Y.-F.; Izawa, T.; Kobayashi, S.; Ohno, M. J. Am. Chem. Soc. 1982, 104,
6465–6466; (b) Kotani, H.; Kuze, Y.; Uchida, S.; Miyabe, T.; Iimori, T.; Okano, K.;
Kobayashi, S.; Ohno, M. Agric. Biol. Chem. 1983, 47, 1363–1365; (c) Iida, H.;
Kasahara, K.; Kobayashi, C. J. Am. Chem. Soc. 1986, 108, 4647–4648; (d) Tanner,
D.; Somfai, P. Tetrahedron Lett. 1988, 29, 2373–2376; (e) De Bernardo, S.; Tengi,
J. P.; Sasso, G.; Weigele, M. Tetrahedron Lett. 1988, 29, 4077–4080; (f) Kasahara,
K.; Iida, H.; Kibayashi, C. J. Org. Chem. 1989, 54, 2225–2233; (g) Schimdt, U.;
Sta¨bler, F.; Lieberknecht, A. Synthesis 1992, 482–486; (h) Maycock, C. D.; Barros,
4.1.18. 5-epi-Negamycin TFA salt (2-[(3R,5S)-3,6-diamino-5-hydroxy-
hexanoyl]-1-methylhydrazinoacetic acid) TFA salt 25. 5-epi-Neg-
amycin was prepared from compound 3R,5S-22 using the same
method as we used to prepare (þ)-negamycin.9 Preparative HPLC
was performed using a C18 reverse-phase column (250ꢂ20 mm;
YMC-Pack ODS-AM) with a binary solvent system; a linear gradient
of CH3CN 0–5% in 0.1% aqueous TFA at a flow rate of 6 mL/min,
detected at 222 nm. This gave after purification 5-epi-negamycin as
25
the TFA salt in a 41% yield over the five steps. [
a
]
ꢀ11.5ꢁ (c 0.55,
D
H2O), lit.7i
[a]
D ꢀ9.4ꢁ (c 0.50, H2O); 1H NMR (400 MHz, D2O)
d
4.14–
M. T.; Santos, A. G.; Godinho, L. S.
Tetrahedron Lett. 1
992, 33, 4633–4636; (i)
Masters, J. J.; Hegedus, L. S. J. Org. Chem. 1993, 58, 4547–4554; (j) Socha, D.;
Jurczak, M.; Chmielewski, M. Tetrahedron Lett. 1995, 36, 135–138; (k) Davies, S.
G.; Ichihara, O. Tetrahedron: Asymmetry 1996, 7, 1919–1922; (l) Jain, R. P.;
Williams, R. M. J. Org. Chem. 2002, 67, 6361–6365; (m) Naidu, S. V.; Kumar, P.
Tetrahedron Lett. 2007, 48, 3793–3796.
8. (a) Chimielewski, M.; Jurczak, J.; Zamoiski, A. Tetrahedron 1978, 34, 2977–2981;
(b) Jang, J.; Lee, Y.; Ahn, Y. Bull. Korean Chem. Soc. 1997, 18, 254–256; (c) Shimizu,
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4.08 (m, 1H), 3.92–3.85 (m, 1H), 3.67 (s, 2H), 3.20 (dd, J¼3.2 and
13 Hz, 1H), 3.01 (dd, J¼8.4 and 13 Hz, 1H), 2.79–2.69 (m, 4H, in-
cluding 3H at
d
2.72, s), 2.62 (dd, J¼7.2 and 16 Hz, 1H), 1.97–1.84 (m,
2H); 13C NMR (100 MHz, D2O)
d 173.7, 169.1, 65.3, 58.9, 46.9, 44.5,
44.2, 35.6, 35.0; HRMS (FABþ) calcd for C9H21N4O4 (MþþH)
249.1563, found 249.1559.
9. Hayashi, Y.; Regnier, T.; Nishiguchi, S.; Sydnes, M. O.; Hashimoto, D.; Hasegawa,
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(a) Takano, S.; Yanase, M.; Sekiguchi, Y.; Ogasawara, K. Tetrahedron Lett. 1987,
28, 1783–1784; (b) Braun, M.; Waldmu¨ller, D. Synthesis 1989, 856–858;
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Acknowledgements
This research was supported by a Research Grant (17A–10) for
Nervous and Mental Disorders from the Ministry of Health, Labour
and Welfare. The authors would also like to thank Mr. Kei Asai and
Ms. Atsuko Ohta, Department of Medicinal Chemistry and Dr.
Minoru Ozeki, Pharmaceutical Manufacturing Chemistry, Kyoto
Pharmaceutical University for conducting some preliminary ex-
periments and for technical guidance of asymmetric Michael
addition.
Supplementary data
Supplementary data including 1H and/or 13C NMR charts of
compounds 8–11, 13, 14, 17–20, 3R,5S-22, 24, 25, 27 and 28. Sup-
plementary data associated with this article can be found in the
online version, at doi:10.1016/j.tet.2009.10.097.