Synthesis and DNA cleavage evaluation of epoxypiperidine derivatives bearing a dehydroamino acid unit

Article abstract of DOI:10.1016/j.tet.2010.08.027

Epoxypiperidine derivatives bearing a dehydroamino acid unit were designed and synthesized as novel DNA alkylating agents based on the structure of azinomycins. A relaxation assay of plasmid DNA revealed that the epoxypiperidine derivative 3 has a DNA cle

Full text of DOI:10.1016/j.tet.2010.08.027

Tetrahedron 66 (2010) 8181e8188
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and DNA cleavage evaluation of epoxypiperidine derivatives bearing
a dehydroamino acid unit
,b,
Takao Yamaguchi a, Yuji Kawada a, Satoshi Obika a, Takeshi Imanishi a, Kazuyuki Miyashita a
*
a Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
b Faculty of Pharmacy, Osaka Ohtani University, 3-11-1, Nishikiori-kita, Tondabayashi,Osaka 584-8540, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 March 2010
Received in revised form 7 August 2010
Accepted 10 August 2010
Available online 14 August 2010
Epoxypiperidine derivatives bearing a dehydroamino acid unit were designed and synthesized as novel
DNA alkylating agents based on the structure of azinomycins. A relaxation assay of plasmid DNA revealed
that the epoxypiperidine derivative 3 has a DNA cleavage activity. Based on the studies, it would appear
that the electron density of the amino group of epoxypiperidines plays a critically important role in the
DNA cleavage.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Epoxypiperidine
Dehydroamino acid
DNA cleavage
DNA alkylation
Azinomycin
1. Introduction
We are interested in azinomycins because of their remarkable
biological activities and peculiar structure, the highly unstable
DNA alkylating agents comprise an extremely important class of
clinical therapeutics in the treatment of cancer, a large number of
which have been discovered from natural products and their syn-
thetic analogs.1 Azinomycins A and B, isolated from Streptomyces sp.
as antitumor antibiotics in 1986, present a new structural class of
dual DNA alkylating (interstrand cross-linking) agents (Fig.1).2 As is
obvious from the structure, the aziridine and oxirane rings are
known to be responsible for the alkylation of a guanosine and/or
adenosine residue of DNA duplex.3
4-hydroxy-1-azabicyclo[3.1.0]hexane ring system.4 We previously
designed and synthesized 3,4-epoxypiperidine derivatives as novel
DNA alkylating agents based on a hypothetical interconversion of
the 3,4-epoxypiperidine and 4-hydroxy-1-azabicyclo[3.1.0]hexane
(Fig. 2).5 As was anticipated, 3,4-epoxypiperidines 1 and 2 showed
DNA cleavage activity at concentrations of 100 and 10
mM, re-
spectively, in a relaxation assay of plasmid DNA. Our studies further
revealed that both the amino group and epoxide are important for
DNA cleavage. Although the mechanism of action is yet to be
DNA
?
NH
N
O
O
O
N
X
H
N
MeO
O
HO
O
N
H
O
O
AcO
O
H
N
DNA
HO
R
ArCH2O
NH
O
azinomycin A: X = H2
AcO
NH
azinomycin B (carzinophilin): X = CHOH
O
O
Figure 1. Structures of azinomycins A and B.
1: Ar = Ph
2: Ar = 2-Nap
3: R = pyrrolidinyl
4: R = OMe
* Corresponding author. Tel.: þ81 721 24 9652; e-mail address: miyasik@
Figure 2. Design of epoxypiperidine derivatives 1e4 based on the hypothetical in-
terconversion of the 3,4-epoxypiperidine and 4-hydroxy-1-azabicyclo[3.1.0]hexane.
osaka-ohtani.ac.jp (K. Miyashita).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.08.027

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T. Yamaguchi et al. / Tetrahedron 66 (2010) 8181e8188
known, 3,4-epoxypiperidine might be a stable precursor of 4-hy-
droxy-1-azabicyclo[3.1.0]hexane. As part of our continuous effort to
study the structureeactivity relationship of the epoxypiperidines,
we report herein, the synthesis and evaluation of the 3,4-epoxy-
piperidines bearing a dehydroamino acid unit.
OH
TBDMSO
O
O
O
O
a
b
HO
OH
O
O
NHBn
NHBn
O
OH
10
OH
11
9
From the structure of azinomycins, it is interestingto decipherhow
the attachment of the unique dehydroamino acid moiety affects the
activity of the epoxypiperidines. It is expected that, in azinomycins,
conjugation of the aziridine ring with the unsaturated amide moiety
would enhance the aziridine-ring cleavage by a DNA nucleophile as
indicated by red arrows in Figure 1.6 We first sought to address this
curiosity by designing compounds 3 and 4 (Fig. 2). As the naphthyl
group (Nap) of 2 and azinomycin appears to be also an important
component probably acting as an interaction site with DNA,5a we in-
troduced it into the dehydroamino acid unit. The synthetic strategy of
3 and 4 is showninFigure 3. We planned to synthesize the compounds
3 and 4 through ring-opening reactions of azlactone 5 by using pyr-
rolidine and MeOH, respectively. The azlactone 5 would be obtained
by a coupling reaction of 1-naphtyl azlactone 6 and thioimidate 7.
c
OH
TBDMSO
O
O
f
NHAlloc
NHR1
O
O
OAc
15
OR2
12: R1, R2 = H
d
e
13: R1 = Alloc, R2 = H
14: R1 = Alloc, R2 = Ac
g
O
S
SMe
N
O
O
O
O
O
i
j
NR
NH
O
OAc
16: R = Alloc
17: R = H
OAc
18
OAc
7
1-Nap
O
h
1-Nap
H2N COOH
glycine
N
O
O
Scheme 2. Synthesis of the thioimidate 7. Reagents and conditions: (a) BnNH2, MeOH,
50 ꢀC, then H2, PtO2, MeOH, 50 ꢀC, 63%; (b) TBDMSCl, imidazole, DMF, 0 ꢀC, 86%; (c)
20 wt.% Pd(OH)2/C, HCO2NH4, EtOH, reflux; (d) AllocCl, Na2CO3, MeOH, H2O, 0 ꢀC; (e)
Ac2O, Py, 0 ꢀC; (f) TBAF, AcOH, THF, 0 ꢀC to rt, quant. (four steps); (g) PCC, MS3 Å,
CH2Cl2, rt; (h) Pd(PPh3)4, sodium 2-methylhexanoate, CH2Cl2, rt, 68% (two steps); (i)
Lawesson’s reagent, toluene, 65 ꢀC, 84%; (j) MeOTf, CH2Cl2, 0 ꢀC, 97%.
N
6
O
3 or 4
+
O
O
NH
SMe
O
O
O
N
HO
OH
OAc
5
HO
OH
-arabinose
OAc
7
D
1-Nap
1-Nap
O
O
Figure 3. Retrosynthetic analysis for the 3,4-epoxypiperidine derivatives 3 and 4.
N
SMe
N
O
N
O
6
O
O
2. Results and discussion
O
O
NH
toluene, reflux
quant.
Synthesis of the compounds, 3 and 4, was achieved as follows.
1-Naphthyl azlactone 6 was readily prepared from glycine in 88%
via an intramolecular condensation of 8 by treatment with DCC in
DMF (Scheme 1).
OAc
7
OAc
5 (E/Z = 2:1)
Scheme 3. Coupling reaction of the 1-naphthyl azlactone 6 and thioimidate 7.
1-Nap
H
N
a
b
O
1-Nap
COOH
glycine
N
O
1-Nap
1-Nap
1-Nap
N
O
O
O
O
3
8
6
N
N
O
H
OH
O
Scheme 1. Preparation of the 1-naphthyl azlactone 6. Reagents and conditions: (a)
H
H
1-naphthoyl chloride, NaOH aq, dioxane, rt; (b) DCC, DMF, 50 ꢀC, 88% (two steps).
3
H
N
O
O
O
O
N
N
O4
O4
The thioimidate 7 was synthesized as shown in Scheme 2. The
starting material 9 was prepared from D-arabinose using a known
5
5
OAc
E-5, H-3: 5.72 ppm
OAc
OAc
Z-5, H-3: 5.99 ppm
procedure.7 Reductive amination of 9 employing a catalytic amount
of Pt2O gave amine 10 in 63%. The amine 10 was converted to al-
cohol 15 in five steps, with an overall yield of 86%. PCC oxidation of
15 directly provided the desired lactam 16. After removal of Alloc
group, thiolactam 18 was obtained by heating with Lawesson’s re-
agent.8 Methylation of 18 using MeOTf afforded the desired thio-
imidate 7 exclusively without production of N-methyl thiolactam.
The coupling reaction of azlactones and thioimidates was
reported by Terashima’s group in 2003.9 Following their method,
compounds 6 and 7 were reacted in toluene under reflux to afford
the coupling product 5 in quantitative yield (Scheme 3). The prod-
uct 5 was obtained as an equilibrium mixture of geometrical isomers
(E/Z¼2:1). The existence of hydrogen bonding between the amine
and the carbonyl group might suggest the favorable formation of the
E isomer (Fig. 4). The isomers could be differentiated by 1H NMR
(CDCl3). That is to say, the H-3 resonance of Z-5 appears at lower field
Figure 4. Equilibrium of the isomers E-5 and Z-5.
Prior to transformation of the azlactone ring to the dehy-
droamino acid system, the piperidine amino group of 5 was pro-
tected by an Alloc group to yield 19. The preferential formation of
the E isomer (E/Z ratio of 19 was 4:1 based on 1H NMR) may be
attributed to the higher stability of the E isomer anion having an
OeNaþ chelation structure. We add that the ratio of E/Z isomers did
not differ after N-Alloc protection. The E/Z isomers were separated
by column chromatography after removal of the acetonide group by
BCl3, to give the desired E-20 in 42% yield. Treatment of E-20 with
pyrrolidine successfully provided the desired dehydroamino acid
21. The regioselective mesylation of 21 took place under typical
mesylation conditions to give 22 as a single compound (72%, two
steps). The regiochemistry of 22 could not be determined spec-
troscopically at this stage, but was confirmed after derivatizing to
(d
5.99 ppm) than that of E-5 (
deshielding effect by the carbonyl group on the azlactone ring.
d 5.72 ppm) due to a magnetic

T. Yamaguchi et al. / Tetrahedron 66 (2010) 8181e8188
8183
24. The compound 22 was converted into epoxypiperidine 23 by
using K2CO3 in MeOH. Acetylation of 23 gave 24, 1H NMR of which
showed the acetoxymethine hydrogen at 5.69 ppm. This fact
strongly supports that the mesylation of 21 exclusively proceeded
at the C-4 hydroxyl group. Finally, removal of the Alloc group of 24
afforded the desired epoxypiperidine 3 as a 1:1 equilibrium mix-
ture of E/Z isomers. The isomeric mixture was then used for DNA
cleavage assay without further separation (Scheme 4).
of 28 was removed at the final step, and the methyl ester 4 was
obtained as a single isomer. 1H NMR study of 4 revealed that the
H-3 has NOE correlation with the aromatic hydrogen. Therefore, the
methyl ester 4 has an E-configuration. The E isomer is considered to
be stable due to an intramolecular hydrogen bonding between the
amino group and methyl ester.
The epoxypiperidines 3 and 4 were then evaluated by a relaxation
assay of supercoiled plasmid DNA as in the previous report.5 Super-
coiled plasmid DNA (form I) metamorphoses into an open circular
DNA (form II, relaxed DNA) upon DNA damage, and this meta-
morphosis can be analyzed by agarose gel electrophoresis. Each
epoxypiperidine was incubated with supercoiled plasmid DNA at
37 ꢀC for 24 h prior to analysis. The results of the DNA cleavage assay
are shown in Figure 5.
1-Nap
O
O
O
N
N
N
N
O
O
Na
O
O
O
a
Na
O
O
O
O
NH
N
OAc
5 (E/Z = 2:1)
OAc
OAc
lane
1
2
3
4
5
6
7
compound
-
3
3
3
3
4
4
(conc./mM)
(-)
(1.0) (2.5) (5.0) (10) (1.0) (10)
1-Nap
O
H
N
O
1-Nap
N
N
O
c
Form II
Form I
O
HO
R1O
R2O
3
NAlloc
NAlloc
X 4
5
OAc
21: X = OH
22: X = OMs
OAc
Figure 5. DNA cleavage activity for the epoxypiperidines 3 and 4. Form I: supercoiled
pBR 322 DNA; Form II: relaxed DNA.
19 (E/Z = 4:1): R1, R2 = CH(CH3)2
E-20: R1, R2 = H
d
b
As can be seen from the gel, the DNA form I was clearly con-
verted into form II at 5.0 mM concentration of 3 (lane 4). Increasing
the concentration of 3 to 10 mM led to the disappearance of both
bands (lane 5), presumably due to critical cleavage of DNA resulting
to fragmentation. On the other hand, no significant activity was
observed for epoxypiperidine 4 up to 10 mM concentration.
The compound 3 was evaluated as a mixture of geometrical iso-
mers. However, we point out that the stereochemistry about the
double bond would not be a factor of the activity, as the double bond
could easily isomerize via iminoeenol intermediate 29 (Fig. 6). On
e
O
O
H
H
N
1-Nap
N
N
N
g
O
O
3
RO
H
NAlloc
NH
AcO
4
5
O
O
23: R = H
3 (E/Z = 1:1)
f
24: R = Ac
Scheme 4. Preparation of the pyrrolidine amide 3. Reagents and conditions: (a) Al-
locCl, NaH, THF, 0 ꢀC, 67%, (recovery of SM, 25%, E/Z¼2:1); (b) BCl3, CH2Cl2, ꢁ78 ꢀC, 42%,
(Z-20, 14%; recovery of SM, 42%, E/Z¼4:1); (c) pyrrolidine, toluene, 0 ꢀC; (d) MsCl, Py,
R
N
R
N
R
N
H
N
H
N
H
N
0
ꢀC, 72% (two steps); (e) K2CO3, MeOH, 0 ꢀC to 10 ꢀC, 58%; (f) Ac2O, Py, rt; (g) Pd
O
H
O
H
O
(PPh3)4, HCO2NH4, CH2Cl2, 0 ꢀC, 82% (two steps).
Step 1 AcO
AcO
AcO
HO
The methyl ester derivative 4 was similarly prepared from diol
E-20 (Scheme 5). In contrast to the pyrrolidine amide 24, isomeri-
zation of the double bond was not observed when the Alloc group
O
O
Nu
29
30
3: R = pyrrolidinyl
4: R = OMe
inactive form
active form
1-Nap
O
H
N
O
1-Nap
Step 2
N
OMe
O
a
c
O
R
R
HO
HO
HO
X
NAlloc
NAlloc
H
N
H
N
OH
O
OAc
OAc
AcO
HO
AcO
HO
N
NH
E-20
25: X = OH
b
26: X = OMs
Nu
Nu
O
OMe
O
H
N
H
N
1-Nap
Figure 6. Possible transformations of the epoxypiperidine derivatives 3 and 4.
OMe
e
O
O
H
H
AcO
3
H
N
comparing the activityof 3 with 4, amide 3 showed DNA cleavage, but
ester 4 did not. This observation could be explained by a difference of
electron-withdrawing property between an amide and an ester. The
electron density on the piperidineenitrogen is lowered by the con-
jugated carbonyl group as shown by black arrows on the structure for
3 or 4 in Figure 6.10 As an ester group is more electron-withdrawing
than an amide group, it is thought that, in the case of ester 4, for-
mation of the aziridine ring by intramolecular nucleophilic attack
RO
NAlloc
NOE: 1.2%
4
5
O
O
27: R = H
28: R = Ac
4, H-3: 6.36 ppm
d
Scheme 5. Preparation of the methyl ester 4. Reagents and conditions: (a) MeOH, TEA,
ꢁ20 ꢀC; (b) MsCl, Py, 0 ꢀC, 82% (two steps); (c) K2CO3, MeOH, 0 ꢀC to rt, 44%; (d) Ac2O,
Py, rt; (e) Pd(PPh3)4, HCO2NH4, CH2Cl2, 0 ꢀC, 74% (two steps).

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T. Yamaguchi et al. / Tetrahedron 66 (2010) 8181e8188
shown by red arrows is hindered. That is to say that ester 4 can
transform to an inactive form 29, but cannot transform to an active
form 30, while amide 3 can transform to both forms, 29 and 30.12
Meanwhile, on comparing the activity of 3 with a simple
epoxypiperidine derivative 2, 3 unexpectedly showed lower
7.48e7.58 (3H, m), 7.68 (1H, dd, J¼1, 7.5 Hz), 7.88e7.92 (1H, m), 7.98
(1H, d, J¼7.5 Hz), 8.32e8.35 (1H, m).
4.1.2. 2-(1-Naphthyl)-4H-oxazol-5-one (6). A mixture of 8 (33 g)
and N,N-dicyclohexyl carbodiimide (38 g, 180 mmol) in DMF
(300 mL) was stirred at 50 ꢀC for 6 h. The mixture was then diluted
with Et2O, and washed with water and brine. After drying over
Na2SO4, the organic layer was concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatography
(CHCl3/hexane¼1:1) to afford the product 6 (29 g, 88%, two steps)
as a yellow powder. Compound 6: Mp 109e112 ꢀC. IR nmax (KBr):
3059, 2945, 1816, 1650, 1574, 1509, 1320 cmꢁ1. 1H NMR (270 MHz,
activity (5.0 mM) than did 2 (10
m
M).5a This fact could be at-
tributed to a similar reason with the amide group slightly de-
creasing the nucleophilicity of the proximal piperidineenitrogen
to prevent the formation of the aziridine (step 1) rather than
activation of the aziridine (step 2). Simultaneously, this suggests
that the step 1 might be a rate-determining step. It might be
also considered that the affinity of 3 with DNA is lower than that
of 2 due to the unfavorably arranged naphthyl group of 3. Al-
though the mechanistic issues still remain to be solved, it would
appear, based on our present results, that the electron density
and/or nucleophilicity of the piperidineenitrogen is critical for
DNA cleavage.11
CDCl3)
d
: 4.59 (2H, s), 7.25e7.69 (3H, m), 7.92 (1H, d, J¼8 Hz), 8.05
(1H, d, J¼8 Hz), 8.17 (1H, d, J¼7 Hz), 9.25 (1H, d, J¼9 Hz). 13C NMR
(67.80 MHz, CDCl3) dC: 55.5, 121.4, 124.4, 125.7, 126.4, 128.0, 128.6,
130.0, 130.5, 133.5, 133.6, 163.0, 175.4. MS (FAB) m/z: 212 (MþHþ).
HRMS Calcd for C13H10NO2: 212.0712. Found: 212.0717.
4.1.3. 3,4-O-Isopropylidene-b-D
-ribofuranose (9)7. The compound 9
3. Conclusion
was prepared from D
-arabinose according to a known procedure.7 2-
Methoxypropene (190 mL, 2.0 mol), DrieriteÒ (20 g) and p-tolue-
In summary, we have demonstrated the design, synthesis, and
evaluation of epoxypiperidine derivatives bearing the dehydroamino
acid unitof azinomycins. The newly-designed epoxypiperidines 3 and
4 were successfully prepared in 19 synthetic steps via coupling of 1-
naphthyl azlactone 6 and thioimidate 7. Relaxation assay of super-
coiled plasmid DNA revealed that epoxypiperidine 3 has a DNA
cleavage activity at a concentration of 5.0 mM. Although epoxy-
piperidine 3 was not as active as the simple epoxypiperidines 1 or 2, it
should be noted that the activity is dependent on a variety of factors.
In fact, we recently reported that a proximal aromatic ring to the
epoxypiperidine is a highly important unit for the DNA cleavage
activity.5b Altogether, these results should guide us in our future de-
sign of advanced epoxypiperidine analogs. Further studies on the
mechanism and structureeactivity relationship of epoxypiperidines
are ongoing.
nesulfonic acid monohydrate (350 mg, 2.0 mmol) were added to
a solution of D-arabinose (150 g,1.0 mol) in anhydrous DMF (700 mL)
at 0 ꢀC, and the mixture was stirred at 5 ꢀC for 8 h. Sodium carbonate
(100 g) was added to the mixture, and the resulting mixture was
stirred at 0 ꢀC for 1 h and then filtrated. The filtrate was poured onto
water at 0 ꢀC, and the aqueous solution was washed with CH2Cl2. The
organic layer was then extracted with water, and the combined
aqueous solution was concentrated under reduced pressure. The
crude product was purified by recrystallization (toluene) to afford
the product 9 (146 g, 77%) as a white solid. Compound 9: Mp
86e87 ꢀC (toluene) [lit.7 mp 82e84 ꢀC]. 1H NMR (270 MHz, CDCl3)
d:
1.37 (3H, s), 1.52 (3H, s), 3.80e3.88 (2H, m), 4.11e4.32 (3H, m), 4.56
(3/10H, d, J¼7 Hz), 5.19 (7/10H, d, J¼4 Hz).
4.1.4. (2R,3S,4R)-5-Benzylamino-2,3-isopropylidenedioxypentane-
1,4-diol (10). Benzylamine (95 mL, 0.87 mol) was added to a solution
of 9 (150 g, 0.79 mol) in MeOH (2.0 L), and the mixture was stirred at
50 ꢀC for 6 h. To a suspension of PtO2 (8.0 g) in MeOH (1.0 L) was
added the mixture, and the resulting mixture was further stirred at
50 ꢀC for 19 h under hydrogen atmosphere. After filtration, the fil-
trate was concentrated under reduced pressure, and the residue was
purified by recrystallization (AcOEt/hexane¼1:3) to afford the
4. Experimental
4.1. General
All chemicals from commercial sources were used without pu-
rification unless otherwise stated. All reactions were performed
under an atmosphere of nitrogen unless noted otherwise. Reactions
were monitored by thin-layer chromatography (TLC) plates that
were visualized using UV light and p-anisaldehyde or phospho-
molybdic acid staining, followed by heating. Column chromatog-
raphy was carried out with BW-127ZH, FL-100D, FL-100B,
PSQ-100B, BW-300, FL-60D silica gel purchased from Fuji Silysia. IR
spectra were obtained from KBr plates using a JASCO FT-IR-4200
instrument. NMR spectra, including 1H, 13C, and NOE experiments,
were recorded on a JEOL EX-270, JEOL AL-300 or JEOL LA-500
spectrometer. Proton and Carbon chemical shifts were referenced
to residual solvent peaks or tetramethylsilane (0.00 ppm). High-
resolution mass spectra were measured on a JEOL JMS-600 or JMS-
700 mass spectrometer.
product 10 (140 g, 63%) as colorless crystals. Compound 10: Mp 63 ꢀC
25
(AcOEt/hexane). [
a
]
þ2.25 (c 0.65, CHCl3). IR nmax (KBr): 3320,
D
2982, 1650, 1539, 1059 cmꢁ1. 1H NMR (270 MHz, CDCl3)
d: 1.36 (3H,
s), 1.50 (3H, s), 2.77 (1H, ABX, JAX¼5 Hz, JAB¼12 Hz), 2.88 (1H, ABX,
JAX¼7 Hz, JAB¼12 Hz), 3.72 (1H, ABX, JAX¼4 Hz, JAB¼12 Hz), 3.76e3.87
(2H, m), 3.82 (2H, s), 4.07 (1H, dd, J¼3, 7 Hz), 4.20 (1H, ddd, J¼3, 4,
5 Hz), 7.21e7.38 (5H, m). 13C NMR (67.80 MHz, CDCl3) dC: 25.2, 27.2,
52.3, 53.6, 60.8, 67.3, 77.3, 78.2, 108.2, 127.1, 128.0, 128.4, 139.4. MS
(EI) m/z: 281 (Mþ, 6),120 (100), 91 (100). HRMS Calcd for C15H23NO4:
281.1627. Found: 281.1644.
4.1.5. (2R,3S,4R)-5-N-Benzylamino-1-tert-butyldimethylsiloxy-2,3-
isopropylidenedioxypentan-4-ol (11). Imidazole (17 g, 240 mmol)
and tert-butyldimethylsilyl chloride (24 g, 160 mmol) were added to
a solution of 10 (45 g, 16 mmol) in anhydrous DMF (150 mL) at 0 ꢀC,
and the reaction mixture was stirred at room temperature for 1 h.
Water was added to the mixture, and the resulting mixture was
extracted with AcOEt. The combined organic layer was washed with
brine and dried over Na2SO4. After removal of the solvent, the crude
product was purified by silica gel column chromatography (CHCl3/
4.1.1. N-(1-Naphthoyl)glycine (8). A solution of 1-naphthoyl chlo-
ride (30 mL, 200 mmol) in dioxane (100 mL) was added to a solu-
tion of glycine (15 g, 200 mmol) in 1.0 M NaOH aq (200 mL). The
mixture was stirred at room temperature for 30 min, and then
washed with AcOEt. The aqueous layer was acidified by an addition
of 5.0 M HCl aq. A precipitate was collected by filtration and dried
under vacuum to afford the product 8 (37 g) as a white powder.
Compound 8: Mp 132e134 ꢀC. IR nmax (KBr): 3050, 1729, 1650, 1536,
MeOH¼10:1) to afford the product 11 (54 g, 86%) as a colorless oil.
21
Compound 11: [
a
]
ꢁ8.7 (c 0.95, CHCl3). IR nmax (KBr): 3478, 2930,
D
1400, 1213 cmꢁ1
.
1H NMR (270 MHz, CDCl3)
d
: 4.17 (2H, s),
1459, 1375, 1253, 1216, 1089 cmꢁ1. 1H NMR (270 MHz, CDCl3)
d: 0.07

T. Yamaguchi et al. / Tetrahedron 66 (2010) 8181e8188
8185
(6H, s), 0.89 (9H, s), 1.34 (3H, s), 1.47 (3H, s), 2.76 (1H, ABX, JAX¼5 Hz,
JAB¼12 Hz), 2.83 (1H, ABX, JAX¼7 Hz, JAB¼12 Hz), 3.74 (1H, ABX,
JAX¼4 Hz, JAB¼11 Hz), 3.80 (2H, s), 3.90 (1H, m), 3.92 (1H, ABX,
JAX¼7 Hz, JAB¼11 Hz), 4.08 (1H, dd, J¼3, 7 Hz), 4.13 (1H, ddd, J¼3, 4,
7 Hz), 7.18e7.33 (5H, m). 13C NMR (67.80 MHz, CDCl3) dC: ꢁ5.4, 18.3,
25.2, 25.9, 27.2, 52.4, 53.8, 61.8, 67.6, 77.2, 78.3, 108.0, 126.7, 127.9,
128.2, 140.1. MS (EI) m/z: 395 (Mþ, 12), 380 (25), 338 (77), 120 (100),
91 (80). HRMS Calcd for C21H37NO4Si: 395.2492. Found: 395.2493.
(300 mL) was added acetic acid (46 mL, 0.80 mol) and TBAF solution
(1.0 M in THF, 180 mL, 180 mmol) at 0 ꢀC, and the mixture was
stirred at room temperature for 24 h. The mixture was diluted with
AcOEt, and washed with saturated NaHCO3, water, and brine. After
drying over Na2SO4, the solvent was removed under reduced
pressure. The residue was purified by silica gel column chroma-
tography (CHCl3/MeOH¼1:0 to 75:1) to afford the product 15 (43 g,
22
quant., four steps) as a colorless oil. Compound 15: [
a
]
þ7.10
D
(c 0.87, CHCl3). IR nmax (KBr): 3341, 2986, 1730, 1538, 1375, 1243,
4.1.6. (2R,3S,4R)-5-Amino-1-tert-butyldimethylsiloxy-2,3-iso-
propylidenedioxypentan-4-ol (12). To a solution of 11 (54 g, 140 mol)
inEtOH (500mL)was added 20 wt.%Pd(OH)2/C (20g) and ammonium
formate (90 g,1.4 mol), and the mixture was refluxed for 15 min. After
1048 cmꢁ1. 1H NMR (270 MHz, CDCl3)
d
: 1.36 (3H, s), 1.47 (3H, s),
2.10 (3H, s), 3.36 (1H, ddd, J¼6, 6, 15 Hz), 3.56 (1H, ABXY, JAX¼6 Hz,
JAY¼6 Hz, JAB¼15 Hz), 3.72 (1H, ABXY, JAX¼4 Hz, JAY¼6 Hz,
JAB¼15 Hz), 3.69 (1H, ABX, JAX¼6 Hz, JAB¼12 Hz), 3.74 (1H, ABX,
JAX¼5 Hz, JAB¼12 Hz), 4.20e4.36 (2H, m), 4.56 (2H, d, J¼5 Hz), 5.15
(1H, dd, J¼6, 9 Hz), 5.21 (1H, dd, J¼1, 10 Hz), 5.30 (1H, dd, J¼1,
17 Hz), 5.91 (1H, m). 13C NMR (67.80 MHz, CDCl3) dC: 21.2, 25.4, 27.2,
42.8, 60.8, 65.7, 69.9, 76.0, 77.2, 108.8, 117.6, 132.5, 156.4, 170.3. MS
(FAB) m/z: 318 (MþHþ). HRMS Calcd for C14H24NO7: 318.1553.
Found: 318.1557.
filtration, the filtrate was concentrated under reduced pressure to
21
afford the compound 12 (42 g) as a colorless oil. Compound 12: [a]
ꢁ11.0 (c 1.05, CHCl3). IR nmax (KBr): 3370, 2933, 1254, 1093 cmꢁ1. 1DH
NMR (270 MHz, CDCl3) d: 0.10 (6H, s), 0.90 (9H, s),1.36(3H, s),1.48 (3H,
s), 2.74 (2H, br s), 2.86e3.00 (2H, m), 3.74 (1H, ABX, JAX¼8 Hz,
JAB¼11 Hz), 3.83(1H, m), 3.94(1H, ABX, JAX¼7 Hz, JAB¼11 Hz), 4.10 (1H,
dd, J¼2, 6 Hz), 4.16 (1H, m).13C NMR (67.80 MHz, CDCl3) dC: ꢁ5.5,18.2,
25.0, 25.7, 27.1, 44.9, 61.6, 68.9, 77.2, 77.9, 107.9. MS (FAB) m/z: 306
(MþHþ). HRMS Calcd for C14H32NO4Si: 306.2101. Found: 306.2126.
4.1.10. (3S,4S,5R)-5-Acetoxy-N-allyloxycarbonyl-3,4-iso-
propylidenedioxypiperidin-2-one (16). Pyridinium chlorochromate
(92 g, 0.42 mol) and molecular sieves 3 Å (45 g) were added to
a solution of 15 (45 g, 140 mmol) in CH2Cl2 (2.5 L), and the mixture
was stirred at room temperature for 30 h. Water was added to the
mixture, and the resulting mixture was filtrated. The filtrate was
extracted with Et2O, and the combined organic layer was washed
with water and brine. After drying over Na2SO4, the solvent was
4.1.7. (2R,3S,4R)-5-N-Allyloxycarbonylamino-1-tert-butyldimethylsi-
loxy-2,3-isopropylidenedioxypentan-4-ol (13). To a solution of 12
(42 g) in MeOH (150 mL) was added water (600 mL), sodium car-
bonate (25 g, 240 mmol), and allyl chloroformate (20 mL,
190 mmol) at 0 ꢀC, and the mixture was stirred for 30 min. Satu-
rated NaHCO3 was added to the mixture, and the resulting mixture
was stirred at room temperature for 10 min, and then extracted
with AcOEt. The combined organic layer was washed with water
and brine and dried over Na2SO4. The solvent was removed under
vacuum to afford the product 13 (54 g) as a colorless oil. Compound
removed under reduced pressure to afford the product 16 (41 g) as
22
a colorless oil. Compound 16: [
a
]
þ0.26 (c 0.97, CHCl3). IR nmax
D
(KBr): 2937, 1784, 1729, 1458, 1375, 1227, 1055 cmꢁ1
.
1H NMR
(270 MHz, CDCl3) : 1.37 (3H, s), 1.52 (3H, s), 2.06 (3H, s), 3.87 (1H,
d
ABX, JAX¼2 Hz, JAB¼14 Hz), 4.27 (1H, ABXY, JAX¼2 Hz, JAY¼4 Hz,
JAB¼14 Hz), 4.44 (1H, ddd, J¼2, 2, 7 Hz), 4.65 (1H, d, J¼7 Hz), 4.76
(2H, d, J¼6 Hz), 5.02 (1H, m), 5.29 (1H, dd, J¼2, 11 Hz), 5.44 (1H, dd,
J¼2, 17 Hz), 5.96 (1H, m). 13C NMR (67.80 MHz, CDCl3) dC: 20.7, 24.1,
26.0, 43.1, 67.8, 67.9, 75.1, 76.1, 111.1, 118.7, 130.8, 152.9, 165.9, 169.1.
MS (FAB) m/z: 314 (MþHþ). HRMS Calcd for C14H20NO7: 314.1240.
Found: 314.1247.
13: [
a
]
D
21 þ3.69 (c 0.95, CHCl3). IR nmax (KBr): 3737, 2933, 1717, 1533,
1253, 1096 cmꢁ1. 1H NMR (270 MHz, CDCl3)
d
: 0.10 (6H, s), 0.90 (9H,
s), 1.35 (3H, s), 1.48 (3H, s), 3.29 (1H, ABXY, JAX¼4 Hz, JAY¼8 Hz,
JAB¼13 Hz), 3.47 (1H, m), 3.76 (1H, ABX, JAX¼3 Hz, JAB¼11 Hz), 3.92
(1H, m), 3.94 (1H, ABX, JAX¼7 Hz, JAB¼11 Hz), 4.07 (1H, dd, J¼3,
6 Hz), 4.16 (1H, ddd, J¼4, 6, 6 Hz), 4.56 (2H, d, J¼5 Hz), 5.19 (1H, dd,
J¼2, 10 Hz), 5.29 (1H, dd, J¼2, 18 Hz), 5.92 (1H, m). 13C NMR
(67.80 MHz, CDCl3) dC: ꢁ5.4, 18.3, 25.1, 25.8, 27.1, 44.6, 61.3, 65.4,
67.8, 76.9, 77.6,108.2,117.3,132.8,156.3. MS (FAB) m/z: 390 (MþHþ).
HRMS Calcd for C18H36NO6Si: 390.2312. Found: 390.2311.
4.1.11. (3S,4S,5R)-5-Acetoxy-3,4-isopropylidenedioxypiperidin-2-one
(17). Tetrakis(triphenylphosphine) palladium (0) (1.6 g, 1.4 mmol),
triphenylphosphine (14 g, 55 mmol), and sodium 2-methylhex-
anoate (96 g, 0.63 mol) were added to a solution of 16 (41 g) in
anhydrous CH2Cl2 (1.5 L), and the mixture was stirred at room
temperature for 1 h. After dilution with AcOEt, the resulting mix-
ture was washed with water and brine, and dried over Na2SO4. The
solvent was removed under reduced pressure, and the residue was
purified by silica gel column chromatography (CHCl3/MeOH¼1:0 to
4.1.8. (2R,3S,4R)-4-Acetoxy-5-N-allyloxycarbonylamino-1-tert-bu-
tyldimethylsiloxy-2,3-isopropylidenedioxypentane (14). To a solution
of 13 (54 g) in anhydrous pyridine (70 mL) was added 4-(dime-
thylamino)pyridine (0.98 g, 8.0 mmol) and acetic anhydride (18 mL,
190 mol) at 0 ꢀC, and the mixture was stirred for 2 h. Saturated
NaHCO3 was added to the mixture, and the resulting mixture was
extracted with AcOEt. The combined organic layer was washed
with water and brine and dried over Na2SO4. The solvent was re-
moved under vacuum to afford the product 14 (59 g) as a colorless
50:1) to afford the product 17 (22 g, 68%, two steps) as a white solid.
25
Compound 17: Mp 99e100 ꢀC. [
a
]
D
ꢁ62.3 (c 0.56, CHCl3). IR nmax
(KBr): 3228, 2988, 1746, 1685, 1375, 1232, 1058 cmꢁ1
.
1H NMR
(300 MHz, CDCl3) : 1.35 (3H, s), 1.45 (3H, s), 2.05 (3H, s), 3.29 (1H,
d
oil. Compound 14: [
a
]
22 ꢁ23.5 (c 0.99, CHCl3). IR nmax (KBr): 3344,
ABXY, JAX¼2 Hz, JAY¼4 Hz, JAB¼13 Hz), 3.71 (1H, AB, J¼13 Hz), 4.39
(1H, dd, J¼5, 7 Hz), 4.48 (1H, d, J¼7 Hz), 5.05 (1H, m), 7.84 (1H, br s).
13C NMR (75.45 MHz, CDCl3) dC: 20.8, 24.6, 26.6, 40.4, 67.9, 72.9,
74.1, 110.7, 169.4, 169.7. MS (EI) m/z: 230 (Mþ, 100), 214 (77), 172
(68), 169 (59), 130 (49), 112 (48), 84 (31). HRMS Calcd for
C10H16NO5: 230.1028. Found: 230.1028.
D
2932, 1734, 1522, 1373, 1251, 1086 cmꢁ1. 1H NMR (270 MHz, CDCl3)
d: 0.07 (6H, s), 0.89 (9H, s), 1.35 (3H, s), 1.45 (3H, s), 2.09 (3H, s), 3.46
(1H, ABX, JAX¼5 Hz, JAB¼12 Hz), 3.52 (1H, ABX, JAX¼3 Hz,
JAB¼12 Hz), 3.69 (2H, d, J¼5 Hz), 4.16e4.28 (2H, m), 4.56 (2H, d,
J¼5 Hz), 5.16 (1H, m), 5.20 (1H, dd, J¼1, 11 Hz), 5.30 (1H, dd, J¼1,
17 Hz), 5.91 (1H, m). 13C NMR (67.80 MHz, CDCl3) dC: ꢁ5.4, 18.3, 21.1,
25.5, 25.8, 27.2, 42.9, 61.5, 65.5, 69.7, 77.0, 77.2, 108.7, 117.4, 132.7,
156.1, 170.0. MS (FAB) m/z: 454 (MþNaþ). HRMS Calcd for
C20H38NO7Si: 432.2418. Found: 432.2412.
4.1.12. (3S,4S,5R)-5-Acetoxy-3,4-isopropylidenedioxypiperidine-2-
thione (18). Lawesson’s reagent (42 g, 100 mmol) was added to
a solution of 17 (22 g, 90 mmol) in toluene (500 mL), and the
mixture was stirred at 65 ꢀC for 30 min. After dilution with AcOEt,
the mixture was washed with saturated NaHCO3 and brine and
then dried over Na2SO4. The solvent was removed under reduced
4.1.9. (2R,3S,4R)-4-Acetoxy-5-N-allyloxycarbonylamino-2,3-iso-
propylidenedioxypentan-1-ol (15). To a solution of 14 (59 g) in THF

8186
T. Yamaguchi et al. / Tetrahedron 66 (2010) 8181e8188
pressure, and the residue was purified by recrystallization (AcOEt/
5.69e6.03 (1H, m), 5.96 (4/5H, d, J¼7 Hz), 6.24 (1/5H, d, J¼7 Hz),
7.53e7.52 (3H, m), 7.93 (1H, d, J¼8 Hz), 8.06 (1H, d, J¼8 Hz), 8.29
(1H, d, J¼8 Hz), 9.41 (1H, d, J¼8 Hz). MS (FAB) m/z: 507 (MþHþ).
HRMS Calcd for C27H27N2O8: 507.1767. Found: 507.1753.
hexane¼1:1) to afford the product 18 (20 g, 84%) as colorless
26
crystals. Compound 18: Mp 164 ꢀC (AcOEt/n-hexane). [
a
]
ꢁ161.8
D
(c 0.56, CHCl3). IR nmax (KBr): 3269, 2987, 1748, 1544, 1375, 1232,
1139, 1053 cmꢁ1. 1H NMR (300 MHz, CDCl3)
d
: 1.41 (3H, s), 1.50 (3H,
s), 2.10 (3H, s), 3.44 (1H, ABXY, JAX¼2 Hz, JAY¼3 Hz, JAB¼14 Hz), 3.69
(1H, ABXY, JAX¼2 Hz, JAY¼2 Hz, JAB¼13 Hz), 4.39 (1H, ddd, J¼2, 3,
7 Hz), 4.95 (1H, d, J¼7 Hz), 5.09 (1H, d, J¼3 Hz). 13C NMR
(75.45 MHz, CDCl3) dC: 20.9, 24.1, 26.3, 42.8, 67.5, 73.2, 78.0, 110.4,
169.7, 197.8. MS (EI) m/z: 245 (Mþ, 100), 230 (32), 188 (23), 128 (57),
100 (41). Anal. Calcd for C10H15NO4S: C, 48.96; H, 6.16; N, 5.71; S,
13.07. Found: C, 48.97; H, 6.05; N, 5.67; S, 12.96.
4.1.16. 4-[(3R,4S,5R)-5-Acetoxy-N-allyloxycarbonyl-3,4-dihydrox-
ypiperidin-2-ylidene]-2-(1-naphtyl)-4H-oxazol-5-one (20). Boron
trichloride solution (1.0 M in hexane, 20 mL, 20 mmol) was added
dropwise over 10 min to a solution of 19 (1.0 g, 2.0 mmol) in anhy-
drous CH2Cl2 (100 mL) at ꢁ78 ꢀC. After stirring for 30 min at ꢁ78 ꢀC,
saturated NaHCO3 solution was added to the reaction mixture,
which was gradually warmed up to room temperature. The resulting
mixture was extracted with AcOEt, and the combined organic layer
was washed with water and brine. After drying over Na2SO4, the
solvent was removed under reduced pressure, and the residue was
purified by silica gel column chromatography (CH2Cl2/MeOH¼50:1)
to afford the product E-20 (390 mg, 42%) as a yellow powder and Z-
20 (131 mg, 14%) as a yellow powder. The starting 19 was recovered
in 42% (420 mg, E/Z¼4:1). Compound E-20: Mp 66e68 ꢀC. IR nmax
(KBr): 3412, 2939, 1796, 1728, 1659, 1565, 1512, 1315, 1231, 1144,
4.1.13. (3S,4S,5R)-5-Acetoxy-3,4-isopropylidenedioxy-2-methylthio-
3,4,5,6-tetrahydropyridine (7). Methyl trifluoromethanesulfonate
(8.9 mL, 80 mmol) was added to a solution of 18 (13 g, 53 mmol) in
anhydrous CH2Cl2 (500 mL) at 0 ꢀC, and the mixture was stirred for
2 h at the same temperature. After an addition of saturated NaHCO3,
the mixture was extracted with AcOEt. The combined organic layer
was washed with water and brine and dried over Na2SO4. The
solvent was removed under vacuum to afford the product 7 (13 g,
1055 cmꢁ1. 1H NMR (270 MHz, CDCl3)
d: 2.12 (3H, s), 3.28 (1H, s),
25
97%) as a white solid. Compound 7: Mp 35ꢁ37 ꢀC. [
a
]
ꢁ36.6 (c
3.75e4.40 (3H, m), 4.57e4.80 (2H, m), 5.08e5.35 (4H, m), 5.92 (1H,
m), 7.52e7.73 (3H, m), 7.95 (1H, d, J¼8 Hz), 8.09 (1H, d, J¼8 Hz), 8.25
(1H, dd, J¼1, 7 Hz), 8.99 (1/2H, d, J¼9 Hz), 9.06 (1/2H, d, J¼8 Hz). MS
(FAB) m/z: 467 (MþHþ). HRMS Calcd for C24H23N2O8: 467.1454.
Found: 467.1443. Compound Z-20: Mp 71e73 ꢀC. IR nmax (KBr): 3425,
2930, 1790, 1727, 1659, 1567, 1512, 1374, 1234, 1141, 1060 cmꢁ1. 1H
D
0.21, CHCl3). IR nmax (KBr): 2930, 1747, 1627, 1373, 1234, 1163,
1058 cmꢁ1. 1H NMR (300 MHz, CDCl3)
d: 1.40 (3H, s), 1.45 (3H, s),
2.08 (3H, s), 2.33 (3H, s), 3.72 (1H, ABX, JAX¼5 Hz, JAB¼16 Hz), 3.86
(1H, ABX, JAX¼3 Hz, JAB¼16 Hz), 4.35 (1H, dd, J¼4, 6 Hz), 4.43 (1H, d,
J¼6 Hz), 5.02 (1H, m). 13C NMR (75.45 MHz, CDCl3) dC: 12.0, 21.0,
25.4, 26.8, 49.3, 68.5, 72.1, 73.4, 110.8, 166.9, 169.9. MS (EI) m/z: 259
(Mþ, 39), 199 (86), 186 (82), 142 (100), 126 (73). HRMS Calcd for
C11H17NO4S: 259.0878. Found: 259.0911.
NMR (270 MHz, CDCl3)
d
: 2.14 (3H, s), 3.12 (1H, d, J¼6 Hz), 3.58 (1H,
ABX, JAX¼7 Hz, JAB¼13 Hz), 3.98 (1H, ddd, J¼4, 7, 7 Hz), 4.35 (1H, ABX,
JAX¼4 Hz, JAB¼13 Hz), 4.65 (1H, m), 4.99 (1H, dd, J¼1,10 Hz), 5.09 (1H,
ddd, J¼1, 3,17Hz), 5.32 (1H, ddd, J¼5, 7, 7Hz), 5.56 (1H, dd, J¼3, 3 Hz),
7.58 (2H, dd, J¼8, 15 Hz), 7.66 (1H, ddd, J¼1, 8, 16 Hz), 7.93 (1H, dd,
J¼1, 8 Hz), 8.08 (1H, d, J¼8 Hz), 8.28 (1H, dd, J¼1, 7 Hz), 9.31 (1H, d,
J¼8 Hz). HRMS Calcd for C24H23N2O8: 467.1454. Found: 467.1445.
4.1.14. 4-[(3R,4S,5R)-5-Acetoxy-3,4-isopropylidenedioxypiperidin-2-
ylidene]-2-(1-naphtyl)-4H-oxazol-5-one (5). The compound 6 (25 g,
54 mmol) was added to a solution of 7 (3.5 g,14 mmol) in anhydrous
toluene (40 mL), and the mixture was stirred at 100 ꢀC for 36 h. The
solvent was removed under reduced pressure, and the residue was
purified by silica gel column chromatography (AcOEt/hexane¼1:6 to
1:4) to afford the product 5 (5.0 g, quant., E/Z¼2:1) as a yellow
powder. Compound 5: Mp 86e88 ꢀC. IR nmax (KBr): 3308, 2987, 2927,
1745, 1712, 1634, 1590, 1509, 1342, 1212, 1160, 1063 cmꢁ1. 1H NMR
4.1.17. (3R,4S,5R)-5-Acetoxy-N-allyloxycarbonyl-2-[(E)-1-(1-pyrroli-
dinyl)carbonyl-1-(1-naphthoyl)amino]methylidene-3,4-dihydrox-
ypiperidine (21). Pyrrolidine (73
mL, 0.88 mmol) was added to
a solution of E-20 (400 mg, 0.88 mmol) in anhydrous THF (10 mL) at
ꢁ20 ꢀC, and the mixture was stirred for 15 min at the same tem-
perature. The solvent was removed under reduced pressure to afford
the product 21 (450 mg) as a colorless amorphous solid. Compound
58b: Mp 218ꢁ220 ꢀC. IR nmax (KBr): 3379, 2977, 2881,1742,1715,1651,
(300 MHz, CDCl3) d: 1.46 (3H, s),1.48 (3H, s), 2.00 (3H, s), 3.57 (1H, m),
3.80 (1H, m), 4.37 (1/3H, ddd, J¼2, 2, 7 Hz), 4.45 (2/3H, ddd, J¼2, 2,
7 Hz), 5.02 (1H, s), 5.72 (2/3H, d, J¼7 Hz), 5.99 (1/3H, dd, J¼2, 7 Hz),
7.42e7.63 (3H, m), 7.80e7.90 (2H, m), 8.08 (1H, m), 9.28 (1H, d,
J¼6 Hz). MS (FAB) m/z: 445 (MþNaþ). HRMS Calcd for C23H22N2O6Na:
445.1376. Found: 445.1377.
1619, 1453, 1399, 1238, 1051 cmꢁ1. 1H NMR (270 MHz, CDCl3)
d: 1.87
(4H, br s), 2.06 (3H, s), 3.33 (4H, br s), 3.68 (1H, br s), 3.87 (1H, br s),
4.23 (1H, br s), 4.49 (2H, s), 4.82 (1H, br s), 5.09 (1H, br s), 5.15 (1H, d,
J¼11 Hz), 5.25 (1H, d, J¼17 Hz), 5.82 (1H, br s), 7.35e7.59 (3H, m), 7.85
(2H, d, J¼8 Hz), 7.93 (1H, d, J¼8 Hz), 8.40 (1H, d, J¼7 Hz). HRMS Calcd
for C28H32N3O8: 538.2189. Found: 538.2195.
4.1.15. 4-[(3R,4S,5R)-5-Acetoxy-N-allyloxycarbonyl-3,4-iso-
propylidenedioxypiperidin-2-ylidene]-2-(1-naphtyl)-4H-oxazol-5-
one (19). Sodium hydride (60% in oil, 610 mg, 15 mmol) was added
to a solution of 5 (3.2 g, 7.6 mmol) in anhydrous THF (70 mL) at 0 ꢀC,
and the mixture was stirred for 30 min at the same temperature.
After an addition of allyl chloroformate (1.5 mL, 11 mmol), the
resulting mixture was further stirred at room temperature for 15 h.
Saturated NaHCO3 was added to the mixture, and the resulting
mixture was extracted with AcOEt. The combined organic layer was
washed with water and brine and dried over Na2SO4. The solvent
was removed under reduced pressure, and the residue was purified
by silica gel column chromatography (AcOEt/hexane¼1:3) to afford
the product 19 (2.5 g, 67%, E/Z¼4:1) as a yellow powder. The
starting 5 was recovered in 25% (0.9 g, E/Z¼2:1). Compound 19: Mp
65e66 ꢀC. IR nmax (KBr): 3023, 2987, 1790, 1751, 1720, 1652, 1512,
4.1.18. (3R,4S,5R)-5-Acetoxy-N-allyloxycarbonyl-3-hydroxy-2-[(E)-
1-(1-pyrrolidinyl)carbonyl-1-(1-naphthoyl)amino]methylidene-4-
methanesulfonyloxypiperidine
(22). Methanesulfonyl
chloride
(0.12 mL, 1.7 mmol) was added to a solution of 21 (450 mg) in
anhydrous pyridine (10 mL) at 0 ꢀC, and the mixture was stirred at
the same temperature for 2 h. The mixture was then added NaHCO3
and extracted with AcOEt. The combined organic layer was washed
with water and brine and dried over Na2SO4. The solvent was re-
moved under reduced pressure, and the residue was purified by
silica gel column chromatography (AcOEt) to afford the product 22
(380 mg, 72%, two steps) as a white powder. Compound 22: Mp
111ꢁ119 ꢀC. [
a
]
22 þ125.2 (c 0.30, CHCl3). IR nmax (KBr): 3248, 2976,
D
1369, 1218, 1056 cmꢁ1. 1H NMR (270 MHz, CDCl3)
d: 1.48 (3/5H, s),
2877, 1747, 1715, 1666, 1611, 1512, 1452, 1396, 1359, 1291, 1229, 1177,
1.51 (12/5H, s), 1.56 (3/5H, s), 1.58 (12/5H, s), 1.96 (3H, s), 3.91 (4/5H,
AB, J¼14 Hz), (1/4H, ABX, JAX¼2 Hz, JAB¼14 Hz), 4.15e4.28 (1H, m),
4.44e4.53 (1H, m), 4.56e4.82 (2H, m), 4.92e5.45 (3H, m),
1139, 1099, 1056 cmꢁ1. 1H NMR (270 MHz, CDCl3)
d: 1.87 (4H, br s),
2.04 (3H, s), 3.12 (3H, s), 3.15e3.57 (4H, m), 3.79 (1H, br s), 4.06 (2H,
br s), 4.49 (1H, br s), 5.13e5.26 (3H, br s), 5.32 (1H, br s), 5.70 (1H, br

T. Yamaguchi et al. / Tetrahedron 66 (2010) 8181e8188
8187
s), 7.35e7.67 (3H, m), 7.75 (1H, d, J¼7 Hz), 7.83 (1H, d, J¼8 Hz), 7.90
(1H, d, J¼8 Hz), 8.33 (1H, d, J¼8 Hz), 9.54 (1H, br s). HRMS Calcd for
C29H34N3O10S: 616.1965. Found: 616.1978.
a solution of E-20 (220 mg, 0.46 mmol) in MeOH (4.6 mL) at ꢁ20 ꢀC,
and the mixture was stirred for 10 min at the same temperature.
After an addition of AcOEt, the mixture was washed with water and
brine and dried over Na2SO4. The solvent was removed under re-
duced pressure to afford the product 25 (230 mg) as a white
powder. Compound 25: Mp 79e85 ꢀC. IR nmax (KBr): 3294, 2951,
1736, 1512, 1493, 1406, 1316, 1238, 1053 cmꢁ1. 1H NMR (270 MHz,
4.1.19. (3R,4R,5R)-N-Allyloxycarbonyl-3-hydroxy-2-[(E)-1-(1-pyrro-
lidinyl)carbonyl-1-(1-naphthoyl)amino]methylidene-4,5-epoxypiper-
idine (23). Potassium carbonate (110 mg, 0.80 mmol) was added to
a solution of 22 (240 mg, 0.40 mmol) in MeOH (4.0 mL) at 0 ꢀC. The
mixture was stirred at 0 ꢀC for 30 min, and then at 10 ꢀC for 4 h.
After an addition of AcOEt, the mixture was washed with water and
brine and dried over Na2SO4. The solvent was removed under re-
duced pressure, and the residue was purified by silica gel column
chromatography (AcOEt) to afford the product 23 (110 mg, 58%) as
CDCl3) d: 2.13 (3H, s), 2.84 (1H, br s), 3.63e3.84 (2H, m), 3.79 (3H, s),
4.50e4.76 (3H, m), 4.82 (1H, d, J¼4 Hz), 5.12e5.40 (3H, m), 5.86
(1H, m), 7.46e7.70 (3H, m), 7.84 (1H, d, J¼7 Hz), 7.92 (1H, d, J¼8 Hz),
8.02 (1H, d, J¼8 Hz), 8.38 (1H, d, J¼6 Hz). HRMS Calcd for
C25H27N2O9: 499.1717. Found: 499.1725.
a white powder. Compound 23: Mp 89e95 ꢀC. [
a]
23 þ211.7 (c 0.27,
4.1.23. (3R,4S,5R)-5-Acetoxy-N-allyloxycarbonyl-3-hydroxy-2-[(E)-
1-methoxycarbonyl-1-(1-naphthoyl)amino]methylidene-4-meth-
anesulfonyloxypiperidine (26). Methanesulfonyl chloride (47 mL,
D
CHCl3). IR nmax (KBr): 3267, 2980, 2877, 1712, 1668, 1649, 1615, 1512,
1449, 1397, 1337, 1289, 1259, 1223, 1133, 1065, 1045 cmꢁ1. 1H NMR
(270 MHz, CDCl3)
d
: 1.82 (4H, br s), 2.71 (1H, br s), 3.18e3.53 (5H,
0.69 mmol) was added to a solution of 25 (230 mg) in anhydrous
pyridine (4.5 mL) at 0 ꢀC, and the mixture was stirred at the same
temperature for 1 h. After addition of saturated NaHCO3 solution,
the reaction mixture was extracted with AcOEt, and the combined
organic layers were washed with water and brine. After drying over
Na2SO4, the solvent was removed under reduced pressure, and the
residue was purified by silica gel column chromatography (AcOEt/
hexane¼3:2) to afford the product 26 (220 mg, 82%, two steps) as
m), 3.53e3.93 (2H, m), 4.24e4.46 (1H, m), 4.46e4.77 (2H, m),
5.12e5.36 (3H, m), 5.84 (1H, m), 7.45e7.62 (3H, m), 7.86 (1H, dd,
J¼2, 7 Hz), 7.93 (1H, d, J¼8 Hz), 7.96 (1H, d, J¼8 Hz), 8.51 (1H, d,
J¼9 Hz), 9.59 (1H, br s). HRMS Calcd for C26H28N3O6: 478.1978.
Found: 478.1975.
4.1.20. (3R,4R,5R)-3-Acetoxy-N-allyloxycarbonyl-2-[(E)-1-(1-pyrro-
lidinyl)carbonyl-1-(1-naphthoyl)amino]methylidene-4,5-epoxypiper-
idine (24). Acetic anhydride (48
a solution of 23 (80 mg, 0.17 mmol) in anhydrous pyridine (1.7 mL),
and the mixture was stirred for 2 h. The mixture was added satu-
rated NaHCO3 at 0 ꢀC, and extracted with AcOEt. The combined
organic layer was washed with water and brine and dried over
Na2SO4. The solvent was removed under reduced pressure to afford
a white powder. Compound 26: Mp 95e99 ꢀC. [
a
]
24 þ150.5 (c 0.57,
D
mL, 0.50 mmol) was added to
CHCl3). IR nmax (KBr): 3307, 3022, 2951, 1731, 1666, 1512, 1491, 1437,
1402, 1362, 1318, 1221, 1177, 1141, 1094, 1050 cmꢁ1
.
1H NMR
(270 MHz, CDCl3) : 2.11 (3H, s), 2.91 (1H, br s), 3.09 (3H, s),
d
3.55e3.89 (4H, m), 4.37 (1H, br s), 4.57 (2H, br s), 4.78 (1H, d,
J¼7 Hz), 5.09 (1H, br s), 5.20 (1H, d, J¼9 Hz), 5.31 (1H, dd, J¼1,
17 Hz), 5.50 (1H, br s), 5.85 (1H, m), 7.35e7.70 (3H, m), 7.80 (1H, d,
J¼7 Hz), 7.89 (1H, d, J¼7 Hz), 7.99 (1H, d, J¼8 Hz), 8.36 (1H, d,
J¼8 Hz). HRMS Calcd for C26H29N2O11S: 577.1492. Found: 577.1501.
the product 24 (86 mg) as a white powder. Compound 24: Mp
23
85e91 ꢀC. [
a]
þ300.3 (c 0.14, CHCl3). IR nmax (KBr): 3302, 2974,
D
2878, 1712, 1678, 1642, 1511, 1486, 1436, 1396, 1337, 1256, 1132,
1057 cmꢁ1. 1H NMR (270 MHz, CDCl3)
d: 1.91 (4H, br s), 2.07 (3H, s),
4.1.24. (3R,4R,5R)-N-Allyloxycarbonyl-3-hydroxy-2-[(E)-1-methox-
ycarbonyl-1-(1-naphthoyl)amino]methylidene-4,5-epoxypiperidine
(27). Potassium carbonate (78 mg, 0.57 mmol) was added to a so-
lution of 26 (160 mg, 0.29 mmol) in MeOH (3.0 mL) at 0 ꢀC. The
mixture was stirred at 0 ꢀC for 30 min, and then at 10 ꢀC for 4 h.
After an addition of AcOEt, the mixture was washed with water and
brine and dried over Na2SO4. The solvent was removed under re-
duced pressure, and the residue was purified by silica gel column
2.71 (1H, br s), 3.32e3.72 (5H, m), 3.72e4.00 (2H, m), 4.36e4.68
(3H, m), 5.22 (1H, dd, J¼1, 10 Hz), 5.30 (1H, ddd, J¼1, 3, 17 Hz), 5.69
(1H, s), 5.93 (1H, m), 7.47e7.63 (3H, m), 7.87 (1H, dd, J¼2, 7 Hz), 7.89
(1H, d, J¼7 Hz), 7.98 (1H, d, J¼8 Hz), 8.56 (1H, d, J¼7 Hz), 9.51 (1H,
br s). HRMS Calcd for C28H30N3O7: 520.2084. Found: 520.2101.
4.1.21. (3R,4R,5R)-3-Acetoxy-2-[(E)-1-methoxycarbonyl-1-(1-naph-
thoyl)amino]methylidene-4,5-epoxypiperidine (3). Tetrakis(triphe-
chromatography (AcOEt/hexane¼1:1) to afford the product 27
26
nylphosphine) palladium (0) (20 mg, 17
mmol) and ammonium
(55 mg, 44%) as a white powder. Compound 27: Mp 76e82 ꢀC. [
a]
D
formate (210 mg, 3.4 mmol) were added to a solution of 24 (86 mg)
in anhydrous CH2Cl2 (1.7 mL) at 0 ꢀC, and the mixture was stirred at
the same temperature for 1.5 h. After an addition of AcOEt, the
mixture was washed with water and brine and dried over Na2SO4.
The solvent was removed under reduced pressure, and the residue
was purified by silica gel column chromatography (AcOEt/
hexane¼3:1) to afford the product 3 (60 mg, 82%, two steps, E/
Z¼1:1) as a white foam. Compound 3: IR nmax (KBr): 3289, 3009,
2950, 1745, 1664, 1612, 1511, 1481, 1280, 1220, 1023 cmꢁ1. 1H NMR
þ341.7 (c 0.45, CHCl3). IR nmax (KBr): 3297, 3013, 2951, 1728, 1666,
1512, 1493, 1436, 1403, 1333, 1272, 1213, 1131, 1038 cmꢁ1. 1H NMR
(270 MHz, CDCl3) d: 3.43e3.58 (2H, m), 3.77 (3H, s), 3.93 (1H, AB,
J¼15 Hz), 4.39 (1H, AB, J¼14 Hz), 4.58 (2H, m), 4.99 (1H, s), 5.13 (1H,
s), 5.17 (1H, d, J¼11 Hz), 5.32 (1H, d, J¼17 Hz), 5.84 (1H, m),
7.42e7.67 (3H, m), 7.85 (1H, d, J¼7 Hz), 7.92 (1H, d, J¼8 Hz), 8.02
(1H, d, J¼8 Hz), 8.39 (1H, d, J¼8 Hz). HRMS Calcd for C23H23N2O7:
439.1505. Found: 439.1502.
(500 MHz, CDCl3)
d
: 1.85 (2H, m) 1.93 (2H, m), 2.03 (3/2H, s), 2.08
4.1.25. (3R,4R,5R)-3-Acetoxy-N-allyloxycarbonyl-2-[(E)-1-methox-
ycarbonyl-1-(1-naphthoyl)amino]methylidene-4,5-epoxypiperidine
(28). Acetic anhydride (31 mL, 0.21 mmol) was added to a solution
of 27 (45 mg, 0.10 mmol) in anhydrous pyridine (1.0 mL), and the
mixture was stirred for 4 h. The mixture was added saturated
NaHCO3 at 0 ꢀC, and extracted with AcOEt. The combined organic
layer was washed with water and brine and dried over Na2SO4. The
(3/2H, s), 3.22 (1/2H, d, J¼3 Hz), 3.24 (1/2H, d, J¼4 Hz), 3.30 (1/2H,
d, J¼4 Hz), 3.33 (1/2H, d, J¼5 Hz), 3.38e3.53 (3H, m), 3.73 (1/2H, m),
3.82 (1/2H, m), 3.99 (1/2H, AB, J¼19 Hz), 4.01 (1/2H, AB, J¼18 Hz),
4.22 (1/2H, AB, J¼19 Hz), 4.30 (1/2H, AB, J¼21 Hz), 5.46 (1H, s), 5.59
(1/2H, d, J¼8 Hz), 5.67 (1/2H, d, J¼8 Hz), 7.36e7.53 (3H, m), 7.66 (1/
2H, d, J¼7 Hz), 7.68 (1/2H, d, J¼8 Hz), 7.79 (1H, d, J¼8 Hz), 7.86 (1H,
d, J¼9 Hz), 8.33 (1H, d, J¼8 Hz). HRMS Calcd for C24H26N3O5:
436.1874. Found: 536.1860.
solvent was removed under reduced pressure to afford the product
26
28 (47 mg) as a white powder. Compound 28: Mp 67e73 ꢀC. [
a]
D
þ276.1 (c 0.21, CHCl3). IR nmax (KBr): 3319, 3016, 2945, 1735, 1718,
4.1.22. (3R,4S,5R)-5-Acetoxy-N-allyloxycarbonyl-2-[(E)-1-methox-
ycarbonyl-1-(1-naphthoyl)amino]methylidene-3,4-dihydroxypiper-
idine (25). Triethylamine (0.32 mL, 2.3 mmol) was added to
1674, 1510, 1483, 1435, 1401, 1373, 1337, 1280, 1253, 1230,
1057 cmꢁ1. 1H NMR (270 MHz, CDCl3)
d: 2.07 (3H, s), 3.36 (1H, m),
3.49 (1H, dd, J¼4, 8 Hz), 3.65e4.00 (1H, m), 3.80 (3H, s), 4.33e4.66

8188
T. Yamaguchi et al. / Tetrahedron 66 (2010) 8181e8188
2. For isolation of azinomycins A and B: (a) Nagaoka, K.; Matsumoto, M.; Oono, J.;
Yokoi, K.; Ishizeki, S.; Nakashima, T. J. Antibiot. 1986, 39, 1527; (b) Yokoi, K.;
Nagaoka, K.; Nakashima, T. Chem. Pharm. Bull. 1986, 34, 4554; (c) Ishizeki, S.;
Ohtsuka, M.; Irinoda, K.; Kukita, K.; Nagaoka, K.; Nakashima, T. J. Antibiot. 1987,
40, 60 For isolation of carzinophilin (azinomycin B): (d) Hata, T.; Koga, F.; Sano,
Y.; Kanamori, K.; Matsumae, A.; Sunagawa, R.; Hoshi, T.; Shima, T.; Ito, S.;
Tominaga, S. J. Antibiot. Ser. A 1954, 7, 107; (e) Shimada, N.; Uekusa, M.; Denda,
T.; Ishii, Y.; Iizaki, T.; Sato, Y.; Hatori, T.; Fukui, M.; Sudo, M. J. Antibiot. Ser. A
1955, 8, 67.
(3H, m), 5.17e5.38 (2H, m), 5.66 (1H, d, J¼2 Hz), 5.84 (1H, m),
7.38e7.67 (3H, m), 7.87 (1H, dd, J¼2, 7 Hz), 7.90 (1H, dd, J¼1, 8 Hz),
7.98 (1H, d, J¼8 Hz), 8.58 (1H, dd, J¼1, 8 Hz). 13C NMR (67.80 MHz,
CDCl3) dC: 20.5, 44.2, 50.8, 51.8, 52.6, 66.7, 67.1, 117.8, 118.1, 124.5,
125.6, 126.0, 126.4, 127.4, 128.1, 130.4, 131.4, 131.5, 131.8, 131.9, 133.7,
153.8, 163.0, 167.4, 171.5. HRMS Calcd for C25H25N2O8: 481.1611.
Found: 481.1630.
3. (a) Armstrong, R. W.; Salvati, M. E.; Nguyen, M. J. Am. Chem. Soc. 1992, 114, 3144;
(b) Alcaro, S.; Coleman, R. S. J. Org. Chem. 1998, 63, 4620; (c) Fujiwara, T.; Sato, I.
Tetrahedron Lett. 1999, 40, 315; (d) Hartley, J. A.; Hazrati, A.; Kelland, L. R.;
Khanim, R.; Shipman, M.; Suzenet, F.; Walker, L. F. Angew. Chem., Int. Ed. 2000,
39, 3467; (e) Alcaro, S.; Coleman, R. S. J. Med. Chem. 2000, 43, 2783; (f)
Hodgkinson, T. J.; Shipman, M. Tetrahedron 2001, 57, 4467.
4. (a) Coleman, R. S.; Kong, J.-S. J. Am. Chem. Soc. 1998, 120, 3538; (b) Coleman, R.
S.; Richardson, T. E.; Carpenter, A. J. J. Org. Chem. 1998, 63, 5738; (c) Hashimoto,
M.; Terashima, S. Heterocycles 1998, 47, 59; Total synthesis of azinomycin A: (d)
Coleman, R. S.; Li, J.; Navarro, A. Angew. Chem., Int. Ed. 2001, 40, 1736.
5. (a) Miyashita, K.; Park, M.; Adachi, S.; Seki, S.; Obika, S.; Imanishi, T. Bioorg. Med.
Chem. Lett. 2002, 12, 1075; (b) Kawada, Y.; Kodama, T.; Miyashita, K.; Imanishi,
T.; Obika, S. Heterocycles 2010, 80, 1249.
6. Hashimoto, M.; Matsumoto, M.; Yamada, K.; Terashima, S. Tetrahedron Lett.
1994, 35, 2207.
7. Gelas, J.; Horton, D. Carbohydr. Res. 1975, 45, 181.
8. Pedersen, B. S.; Lawesson, S. O. Tetrahedron 1979, 35, 2433.
9. (a) Hashimoto, M.; Matsumoto, M.; Terashima, S. Tetrahedron 2003, 59, 3019;
(b) Hashimoto, M.; Matsumoto, M.; Terashima, S. Tetrahedron 2003, 59, 3041;
(c) Hashimoto, M.; Sugiura, M.; Terashima, S. Tetrahedron 2003, 59, 3063; (d)
Hashimoto, M.; Matsumoto, M.; Yamada, K.; Terashima, S. Tetrahedron 2003, 59,
3089.
4.1.26. (3R,4R,5R)-3-Acetoxy-2-[(E)-1-methoxycarbonyl-1-(1-naph-
thoyl)amino]methylidene-4,5-epoxypiperidine (4). Tetrakis(triphe-
nylphosphine) palladium (0) (6.0 mg, 5.2 mmol) and ammonium
formate (120 mg, 2.0 mmol) were added to a solution of 28 (47 mg)
in anhydrous CH2Cl2 (1.0 mL) at 0 ꢀC, and the mixture was stirred at
the same temperature for 1 h. After an addition of AcOEt, the
mixture was washed with water and brine and dried over Na2SO4.
The solvent was removed under reduced pressure, and the residue
was purified by silica gel column chromatography (AcOEt/
hexane¼3:2) to afford the product 4 (29 mg, 74%, two steps) as
24
a white powder. Compound 4: Mp 93e95 ꢀC. [
a
]
ꢁ16.1 (c 0.14,
D
CHCl3). IR nmax (KBr): 3289, 3008, 2949, 1745, 1664, 1612, 1511, 1481,
1437, 1369, 1279, 1220, 1070, 1023 cmꢁ1. 1H NMR (500 MHz, CDCl3)
d: 2.15 (3H, s), 3.50 (2H, m), 3.74 (3H, s), 3.86 (2H, s), 6.36 (1H, s),
6.76 (1H, br s), 7.47e7.58 (3H, m), 7.75 (1H, d, J¼8 Hz), 7.86 (1H, dd,
J¼1, 8 Hz), 7.92 (1H, d, J¼9 Hz), 8.37 (1H, d, J¼8 Hz), 8.90 (1H, br s).
HRMS Calcd for C21H21N2O6: 397.1400. Found: 397.1408.
10. Our previous studies revealed that a free piperidine-amino group plays an
important role in DNA cleavage.5a Compound 1 was shown to be more active
than 31, while 32 did not exhibit any activity up to 100 mM. We have examined
the DNA binding of 2 with DNA, but could not obtain clear data probably due to
nonspecific and multi-site binding of 2 with DNA. In addition, NMR analysis did
not show any structural change of 1 or 2 by an addition of DNA.
4.2. Examination of relaxation assay of supercoiled
plasmid DNA5
To a solution of supercoiled pBR 322 DNA (0.15
mg) in pH 7.0 TE
buffer (9
m
L) was added a DMSO solution of compound 3 or 4 (1 mL,
10e100 mM), and the mixture was incubated for 24 h at 37 ꢀC. Under
the concentrations examined, precipitation was not observed.
The resulting DNA analysis was conducted using electrophoresis
BnO
NR
O
(tris-acetateeEDTA buffer, ethidium bromide 1.3 mM solution) on
0.7% native agarose gel at 7.4 v/cm for 30 min.
1: R = H > 31: R = Me > 32: R = CO2Et (inactive)
Supplementary data
11. We also mention that both N-Alloc epoxypiperidines 24 and 28 do not show
any activity up to 10 mM.
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.tet.2010.08.027.
12. The NMR analysis of 3 or 4 shows only one form, and we could observe neither
29 nor 30. Therefore the ratio of these compounds could not be calculated.
However, based on the fact that the double bond of dehydroamino acid de-
rivatives isomerizes via an iminoeenol structure like 29 as shown in Figure 6,
the equilibrium between 3 (or 4) and 29 should exist. In addition, the formation
of 30 was supported by our previous studies, which revealed that a free
piperidine-amino group (electron-density and/or nucleophilicity of piperidine-
amino group) plays an important role in DNA cleavage.5,10,11
References and notes
1. (a) Rajski, S. R.; Williams, R. M. Chem. Rev. 1998, 98, 2723; (b) Wolkenberg, S. E.;
Boger, D. L. Chem. Rev. 2002, 102, 2477.