Syntheses of N-acylisoxazolidine derivatives, related to a partial structure found in zetekitoxin AB, A golden frog poison

Article abstract of DOI:10.3987/COM-08-S(D)44

Syntheses of four N-acylisoxazolidine derivatives related to a partial structure of zetekitoxin AB were described. ¹³C NMR spectra of these compounds could not explain an unusual chemical shift observed in the N-acylisoxazolidine moiety of zete

Full text of DOI:10.3987/COM-08-S(D)44

HETEROCYCLES, Vol. 79, 2009
379
HETEROCYCLES, Vol. 79, 2009, pp. 379 - 385. © The Japan Institute of Heterocyclic Chemistry
Received, 29th September, 2008, Accepted, 28th November, 2008, Published online, 2nd December, 2008.
DOI: 10.3987/COM-08-S(D)44
SYNTHESES OF N-ACYLISOXAZOLIDINE DERIVATIVES, RELATED
TO A PARTIAL STRUCTURE FOUND IN ZETEKITOXIN AB, A
GOLDEN FROG POISON
Toshio Nishikawa,* Daisuke Urabe, and Minoru Isobe
Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho,
Chikusa-ku, Nagoya, 464-8601, Japan. nisikawa@agr.nagoya-u.ac.jp
Abstract – Syntheses of four N-acylisoxazolidine derivatives related to a partial
structure of zetekitoxin AB were described. 13C NMR spectra of these
compounds could not explain an unusual chemical shift observed in the
N-acylisoxazolidine moiety of zetekitoxin.
INTRODUCTION
In 1969, H. Mosher and co-workers reported isolation of zetekitoxin, a water soluble toxic compound
from Panamanian Golden Frog, Atelopus zeteki.1 Pharmacological studies revealed that the biological
profile of zetekitoxin is very similar to tetrodotoxin and saxitoxin, well-known voltage-dependent Na+
channel blocker.2 However, since the frog is designated as an endangered species,3 further collection of
the frog is difficult and the structure remains to be clarified.
In 2004, Yamashita, Daly and co-workers proposed the structure of zetekitoxin AB to be as depicted for
compound 1 (Figure 1) from extensive NMR and MS analyses using about 0.3 mg of the remaining
sample.4 The structure of zetekitoxin is closely related to saxitoxin (2),5 a paralytic shellfish poison, but
possesses two additional unique structural features such as a 10-membered macrolactam fused with
isoxazolidine and a N-hydroxyl carbamate connected to guanidine through a methylene unit.
Interestingly, a carbon signal observed at 156.5 ppm in the 13C NMR spectrum was assigned to the
carbonyl carbon at the C-13 position, a carbonyl carbon of the N-acylisoxazolidine. Since carbonyl
carbon of the N-alkoxyamide is generally observed around 170-175 ppm (ex. a Weinreb amide (3) in
Figure 1), the value of 156.5 ppm is clearly unusual. One possible explanation may be an effect of the
guanidinium groups at α- and β- position of the amide carbonyl group. We found that 13C-NMR
spectrum of bicyclic isoxazolidine 46 showed a chemical shift at 155 ppm for the carbonyl carbon,
suggesting a strong effect of the nitro group at the α-position of the amide.

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HETEROCYCLES, Vol. 79, 2009
156.5 ppm
H
O
O
N
H2N
O
172 ppm
H
N
155 ppm
OH
C13
HN
H
O
H
H
N
N
O
O
HN
O
O
NO2
N
C
NH2
O
N
1
7
1
5
NH2
MeO
C
N
H
H2N
1
6
N
N
H
OH
OH
H2N
N
Me
1
8
14
OH
Ph
HO
11 OH
O
HO3SO
zetekitoxin AB (1)
saxitoxin (2)
Weinreb amide (3)
4
Figure 1. Structures of zetekitoxin AB and its related compounds
In order to find out the reason(s) for the unusual value of the chemical shift observed in zetekitoxin, we
planed to synthesize four compounds 5, 6, 7, and 8 related to the partial structure of zetekitoxin (Figure 2).
13C-NMR spectra of these compounds should clarify the effect of the guanidinium group on the chemical
shift of the carbonyl carbon.
NH2
O
NH2
O
O
O
C
NH
NH2
NH2
O
C
N
C
C
O
O
NH2
NH2
O
N
N
N
HN
HN
OH
HO
5
8
7
6
Figure 2. Structures of model compounds for the N-acylisoxazolidine moiety of zetekitoxin AB (1)
Syntheses of N-acylisoxazolidines
N-Acyl isoxazolidine 57 was prepared by acetylation of the known isoxazolidine hydrochloride 98 (Eq. 1).
O
AcCl, Et3N
O
O
NH·HCl
N
(Eq. 1)
CH2Cl2,
0°C
(92%)
9
5
Synthesis of 6 commenced with a nitro aldol reaction of the known aldehyde 109 with nitromethane in the
presence of KF as a base10 (Scheme 1). The aldol product was dehydrated under conditions using
methanesulfonylation to give nitro olefin 11 in a 53% overall yield from 10. Conjugate reduction was
carried out with NaBH4 in the presence of silica gel11 to afford 12 in high yield. Upon treatment of 12
with Boc2O and DMAP,12 the resulting nitrile oxide 13 underwent an intramolecular 1,3-dioplar
cycloaddition to give a diastereomeric mixture of 14a and 14b in 64 and 22% yield, respectively. After
separation by silica gel column chromatography, the major product 14a was reduced with NaBH3CN in
acetic acid followed by acetylation to give N-acetylisoxazolidine 15. On the other hand, the minor
product 14b was transformed to N-acetylisoxazolidine 16 by similar reactions. The stereochemistries of
15 and 16 were determined by NOESY spectra as shown in Scheme 1. After hydrolysis of the acetonide
of major product 15 with TFA in aqueous methanol, the resulting 1,2-diol was cleaved with NaIO4, and

HETEROCYCLES, Vol. 79, 2009
381
subsequent reduction with NaBH4 afforded a 15:1 mixture of diol 613 and its epimer in a combined yield
of 69%. The stereochemistry of 6 was confirmed by conducting NOESY experiments on
tetrahydropyran 18 derived from 6 under the Mitsunobu condition.
NO2
NO2
1) MeNO2, KF
2-PrOH, rt
Boc2O
DMAP
CHO
NaBH4, SiO2
2) MsCl, Et3N
AcOEt, rt
(53%)
CHCl3, 2-PrOH
(97%)
toluene, rt
O
O
O
O
O
O
10
11
12
O
O N
O N
N
[2+3]
H
O
H
+
O
O
O
O
O
64%
22%
14a
14b
13
1) NaBH4CN, AcOH, rt
2) Ac2O ,Py
(69% in 2 steps)
(55% in 2 steps)
NOESY
O
O
CH3
CH3
H3C
H3C
H
H
O N
O N
O
H
O
H
O
H
O
H
O
H
N
O
N
O
H
H
H
H
H
H
H
O
H
O
O
O
O
NOESY
NOESY
16
15
NOESY
O
O
O
15:1
O N
O N
1) NaIO4
MeOH-H2O
O N
H
H
DEAD, Ph3P
TFA
15
toluene-THF
rt (67%)
H
H
2) NaBH4
2-PrOH
(69%)
MeOH-H2O
(94%)
O
HO
OH
HO
OH
17
6
18
Scheme 1. Synthesis of model compound 6
N-Acyl isoxazolidine 7 was synthesized by coupling isoxazolidine hydrochloride 9 with Fmoc-glycine 19
(Scheme 2). The coupling was carried out by EDCI and HOBt to give amide 20 in 55% yield. The
Fmoc group was deprotected with morpholine and the resulting crude amine was directly treated with
Boc-protected isothiourea (21) in one-pot to afford di-Boc guanidine 2214 in 60% overall yield. The two
Boc groups were then deprotected with TFA in CH2Cl2 to give N-acyl isoxazolidine 715 in 69% yield after
recrystallization from EtOH. NMR spectra of compound 7 was measured in DMSO-d6, since 7 forms
the 5-membered guanidine 23 in D2O via an intramolecular acylation.

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HETEROCYCLES, Vol. 79, 2009
O
i) morpholine
DMF
O
EDCI, HOBt
O
O
NH·HCl
N
+
HO
19
NHFmoc
Et3N
THF, 0 °C
(55%)
SMe
BocHN NBoc (21)
(60%)
ii)
NHFmoc
9
20
O
O
CF3COO
NH2
O
TFA
D2O
O
O
N
N
N
NBoc
HN
HN
CH2Cl2
(69%)
35 °C
HN
NHBoc
NH2
NH2
22
7
23
Scheme 2. Synthesis of model compound 7
N-Acyl isoxazolidine 8 would be synthesized from a commercially available DL-2,3-diaminopropionic
acid monohydrochloride 24. The two amino groups of 24 were protected with Fmoc groups, and the
resulting 25 was coupled with isoxazolidine hydrochloride 9 in the presence of EDCI and HOBt to give
amide 26 in 72% yield. Deprotection of the Fmoc groups was followed by guanidinylation with
Boc-protected isothiourea (21) in the presence of HgCl2 to afford bis-di-Boc guanidine 27.16 However,
the formation of guanidine 8 by deprotection of the four Boc groups under acidic conditions was
unsuccessful, probably due to a similar intramolecular acylation of guanidine as encountered in the
synthesis of 7.
O
O
O
9, EDCI
HOBt, Et3N
NHFmoc
NHFmoc
Fmoc-Cl
NHFmoc
NHFmoc
NH2·HCl
O
N
HO
HO
NH2
Na2CO3 aq.
dioxane
CH2Cl2
(72%)
24
25
26
(64%)
H2N
NH
BocHN
NH
i) piperidine
DMF, 0 °C
O
TFA-CH2Cl2
O
NH2
NHBoc
O
O
N
N
ii) HgCl2, Et3N
SMe
NH2
NHBoc
NHBoc
HN
HN
NH2
(21)
BocHN NBoc
(72%)
27
8
Scheme 3. Synthesis of model compound 8
With the synthetic compounds 5, 6, 7, and 27 in our hands, 13C NMR chemical shifts of the amide
carbonyl group of these compounds were compared with zetekitoxin AB (Table 1). Although the model
13
compound 8 could not be prepared, C NMR spectrum of 27 was used instead of 8 for the comparison,
because the Boc groups of the guanidine have little influence on the chemical shifts of the amide carbon,
judging from comparison between 22 and 7 (Table 1). The model compounds 5 and 22 show two

HETEROCYCLES, Vol. 79, 2009
383
different amide absorptions, respectively, due to the existence of amide rotamers. Chemical shifts for
the amide carbon of 5 and 6 were observed in the usual range for amide carbons. Chemical shifts for the
amide carbon in 7 and 27 were shifted upfield by about 5 ppm due to the inductive effect of guanidine at
the α-position. The guanidine function at β-position has little influence on the amide carbon.
Table 1. 13C NMR chemical shifts for the amide carbonyl carbon of 1, 5, 6, 22, 7, and 27
chemical shift of
amide carbon (ppm)
chemical shift of
amide carbon (ppm)
compound
compound
H
O
O
C13
HN
H2N
O
N
H
N
OH
C
H
O
N
NHBoc
N
O
HN
166.0, 168.3a, c
O
N
NH2
NHBoc
N
H
156.5
22
OH
OH
HO
HO3SO
O
zetekitoxin AB (1)
C
O
N
NH2
NH2
HN
O
168.3b
C
O
N
170.1, 173.3a
7
5
BocHN
NH
NHBoc
NHBoc
NHBoc
O
O
C
C
O
N
O
172.7a
N
167.0b
HN
OH
HO
6
27
a 13C NMR was measured in CDCl3. b 13C NMR was measured in DMSO-d6. c Amide carbons were assigned
from HMBC correlation between the methylene proton on isoxazolidine and the carbonyl carbon.
CONCLUSION
13
We have synthesized N-acyl isoxazolidine derivatives 5, 6, 7 and 27, and measured C NMR spectra of
13
these synthetic compounds. Comparison of the C NMR chemical shift of 5, 6, 7, 27 and zetekitoxin
led us to conclude that the unusual chemical shift of the C-13 of zetekitoxin AB could not be rationalized
only by the substituent effect of the guanidinium groups. The chemical shift at the C-13 might be
explained by unknown factor(s) derived from the rigid 10-membered macrolactam structure of
zetekitoxin. Further efforts to find the factor are in progress in our laboratory.

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ACKNOWLEDGEMENTS
This work was financially supported by PRESTO, JST, a Grand-in Aid for Scientific Research, 21st
century COE program and Global-COE program from MEXT, and the Naito Foundation. We thank
Prof. M. Yotsu-Yamashita (Tohoku University, Japan) and Dr. M. Uchiyama (RIKEN, Japan) for
valuable discussion. We thank Dr. M. Sydnes for valuable help during manuscript preparation.
REFERENCES AND NOTES
1. F. A. Fuhrman, G. J. Fuhrman, and H. S. Mosher, Science, 1969, 165, 1376.
2. G. B. Brown, Y. H. Kim, H. Küntzel, H. S. Mosher, G. J. Fuhrman, and F. A. Fuhrman, Toxicon,
1977, 15, 115.
3. See: The IUCN Red list of Threatened Species, (http://www.iucnredlist.org/static/introduction).
4. M. Yotsu-Yamashita, Y. H. Kim, S. C. Dudley, G. Choudhary, A. Pfahnl, Y. Oshima, and J. W.
Daly, Proc. Nalt. Acad. Sci. USA, 2004, 101, 4346.
5. For a recent review on saxitoxin, see: L. E. Llewellyn, Nat. Prod. Rep., 2006, 23, 200.
6. K. S.-L. Huang, E. H. Lee, M. M. Olmstead, and M. J. Kurth, J. Org. Chem., 2000, 65, 499.
7. Compound (5): 1H NMR (400 MHz, CDCl3) δ 2.32 (2H, quintet J = 7 Hz, CH2-CH2-CH2), 3.71 (2H,
t, J = 7 Hz, N-CH2), 3.97 (2H, t, J = 7 Hz, O-CH2). 13C NMR (75 MHz, CDCl3) δ 20.2, 20.7, 27.5,
42.9, 69.1, 170.7, 173.3. Anal. Calcd for C5H9NO2: C, 52.16; H, 7.88; N, 12.17. Found: C, 52.16;
H, 7.97; N, 12.38.
8. T. L. Cupps, R. H. Boutin, and H. Rapoport, J. Org. Chem., 1985, 50, 3972.
9. H. R. Moon, W. J. Choi, H. O. Kim, and L. S. Jeong, Tetrahedron: Asymmetry, 2002, 13, 1189.
10. (a) R. H. Wollenberg and S. J. Miller, Tetrahedron Lett., 1978, 19, 3219. (b) R. V. Stevens, N.
Beaulieu, W. H. Chan, A. R. Daniewski, T. Takeda, A. Waldner, P. G. Williard, and U. Zutter, J. Am.
Chem. Soc., 1986, 108, 1039.
11. A. K. Sinhababu and R. T. Borchardt, Tetrahedron Lett., 1983, 24, 227.
12. Y. Basel and A. Hassner, Synthesis, 1997, 309.
13. Compound (6): 1H NMR (400 MHz, CDCl3) δ 1.52 (1H, m, CHAHB-CH2-O), 1.86 (1H, dddd, J = 14,
12, 5, 3 Hz, CHAHB-CH2-O), 2.16 (3H, s, Ac), 2.94 (1H, m, -CH-), 3.58 (1H, td, J = 12, 3 Hz,
CH2-CHAHB), 3.69 (1H, m, CH-CHAHB), 3.70 (1H, dd, J = 10.5, 8 Hz, CH-CHAHB-OH), 3.83 (1H,
dd, J = 10.5, 6 Hz, CH-CHAHB-OH), 4.04 (2H, d, J = 6 Hz, CH2-O), 4.67 (1H, ddd, J = 12, 8, 6 Hz,
N-CH). 13C NMR (75 MHz, CDCl3) δ 20.1, 31.2, 46.2, 54.2, 58.6, 59.7, 72.0, 172.7. HRMS
(FAB) for C8H16NO4 (M+H), calcd 190.1079, found 190.1066. Compound (6): 1H NMR (400 MHz,
CDCl3) δ 1.52 (1H, m, CHAHB-CH2-O), 1.86 (1H, dddd, J = 14, 12, 5, 3 Hz, CHAHB-CH2-O), 2.16

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(3H, s, Ac), 2.94 (1H, m, -CH-), 3.58 (1H, td, J = 12, 3 Hz, CH2-CHAHB), 3.69 (1H, m, CH-CHAHB),
3.70 (1H, dd, J = 10.5, 8 Hz, CH-CHAHB-OH), 3.83 (1H, dd, J = 10.5, 6 Hz, CH-CHAHB-OH), 4.04
(2H, d, J = 6 Hz, CH2-O), 4.67 (1H, ddd, J = 12, 8, 6 Hz, N-CH). 13C NMR (75 MHz, CDCl3) δ
20.1, 31.2, 46.2, 54.2, 58.6, 59.7, 72.0, 172.7. HRMS (FAB) for C8H16NO4 (M+H), calcd 190.1079,
found 190.1066.
1
14. Compound (22): H NMR (400 MHz, CDCl3) major peak; δ 1.50 (18H, s, Boc x 2), 2.33 (2H,
quintet J = 7 Hz, CH2-CH2-CH2), 3.75 (2H, t, J = 7.5 Hz, N-CH2), 3.99 (2H, t, J = 7 Hz, O-CH2),
4.34 (2H, d, J = 4.5 Hz, CO-CH2), 9.08 (1H, m, NH), 11.40 (1H, br s, NH). minor peak; δ 1.51
(18H, s, Boc x 2), 3.45 (2H, t, J = 5 Hz, CH2-CH2-CH2), 3.67 (4H, m, CH2-N, CH2-O), 4.25 (2H, d, J
= 4 Hz, CH2-NH), 9.42 (1H, m, NH), 11.39 (1H, m, NH). 13C NMR (75 MHz, CDCl3) δ 27.3, 28.0,
28.2 (two peaks), 42.3, 42.5, 42.9, 43.5, 44.8, 66.3, 66.7, 69.4, 79.3, 79.7, 83.1, 83.3, 152.6, 152.7,
155.4, 155.8, 162.8, 163.1, 166.0, 168.2. Anal. Calcd for C16H28N4O6: C, 51.60; H, 7.58; N, 15.04.
Found: C, 51.60; H, 7.50; N, 14.89.
1
15. Compound (7): H NMR (400 MHz, DMSO-d6) δ 2.26 (2H, quintet, J = 7 Hz, CH2-CH2-CH2), 3.69
(2H, t, J = 7 Hz, N-CH2), 3.97 (2H, t, J = 7 Hz, O-CH2), 4.10 (2H, d, J = 5 Hz, CO-CH2), 7.20-7.72
(5H, m, NH). 13C NMR (100 MHz, DMSO-d6) δ 27.3, 42.4, 43.9, 69.4,157.7, 168.3. HRMS
(FAB) for C6H13N4O2 (M+H), calcd 173.1039, found 173.1078.
1
16. Compound (27): H NMR (400 MHz, DMSO-d6) δ 1.38 (18H, s, Boc x 2), 1.45 (9H, s, Boc), 1.46
(9H, s, Boc), 2.30 (2H, m, CH2CH2CH2), 3.47 (1H, m, N-CHAHB), 3.60-3.82 (3H, m, N-CHAHB,
CH2NH), 4.10 (1H, m, O-CHAHB), 4.17 (1H, m, O-CHAHB), 5.06 (1H, m, CH), 8.42 (1H, br s, NH),
8.76 (1H, br s, NH), 11.41 (1H, br s, NH), 11.45 (1H, br s, NH). 13C NMR (100 MHz, DMSO-d6)
δ 15.3, 25.6, 27.3, 27.7, 27.8, 28.1, 30.6, 31.5, 43.7, 50.9, 51.0, 55.1, 65.1, 69.5, 78.3, 78.4, 78.5,
87.0, 83.1, 83.2, 83.3, 148.5, 151.2, 151.8, 151.9 (two peaks), 155.0, 156.2, 162.8, 162.9, 163.0,
163.1, 167.0. HRMS (FAB) for C28H50N7O10 (M+H), calcd 644.3619, found 644.3593.