An efficient and versatile synthesis of all structural types of acylpolyamine spider toxins

Article abstract of DOI:10.1016/j.tet.2006.06.051

An efficient and versatile synthesis of acylpolyamine spider toxins of all structural types classified by extensive MS analysis has been achieved. By using 2-nitrobenzenesulfonamide as an effective activating and/or protecting group (the Nosyl strategy),

Full text of DOI:10.1016/j.tet.2006.06.051

Tetrahedron 62 (2006) 8335–8350
An efficient and versatile synthesis of all structural types of
acylpolyamine spider toxins
Ken-ichi Nihei,a,y Massuo J. Kato,a Tetsuo Yamaneb and Katsuhiro Konnoc,
*
aInstitute of Chemistry, University of Sa~o Paulo, Sa~o Paulo, SP 05508-900, Brazil
bAmazon Biotechnology Center, Manaus, MA 69075-351, Brazil
cCenter for Applied Toxinology, Butantan Institute, Sa~o Paulo, SP 05503-900, Brazil
Received 24 April 2006; revised 14 June 2006; accepted 14 June 2006
Available online 10 July 2006
Dedicated to Professor T. Okuno on the occasion of his 65th birthday
Abstract—An efficient and versatile synthesis of acylpolyamine spider toxins of all structural types classified by extensive MS analysis has
been achieved. By using 2-nitrobenzenesulfonamide as an effective activating and/or protecting group (the Nosyl strategy), the naturally
occurring toxins 1–8 corresponding to Types A–F were concisely synthesized in high overall yield.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
and the combination of each element results in a complex
mixture of the venom gland constituents as classified into
the generalized structures Types A–F based on the distinct
polyamine backbone structure (Fig. 1).11–13 Figure 2 shows
the representative toxins for each type. However, the rela-
tionships between these structural types and its biological
activities are still not documented well,14 and in the case
of novel toxins, the structures should be confirmed by syn-
thesis.12,15,16 This situation prompted us to establish an effi-
cient and versatile synthetic method for acylpolyamine
toxins. We report herein the successful results along this
line, which enabled us to synthesize eight naturally occur-
ring acylpolyamine spider toxins, JSTX-3 (1), NPTX-8
(2), NPTX-1 (3), NPTX-473 (4), NPTX-501 (5), NSTX-3
(6), joramine (7), and Arg-636 (8) (Fig. 2), covering all
structural types.17
Acylpolyamine toxins, found in spider and wasp venoms,1,2
have attracted much interest in the field of neurobiology
because of its unique activity as open channel blocker of
glutamate receptors.3,4 In order to investigate biological
properties of these toxins in detail, synthesis of a number of
congeners and derivatives is necessary because only limited
quantity is available from natural sources. Therefore, the
acylpolyamine toxins have been an interesting target for
organic synthesis.5–8
Recent developments of mass spectrometric techniques
accelerated and facilitated structural elucidation of acyl-
polyamine spider toxins even at low picomolar levels.9,10
Itagaki et al. revealed that, using highly sensitive analytical
method with LC-MS and MS/MS, the Nephila and Nephi-
lengys spider venom glands contain a complex mixture of
closely related toxins, a majority of which have not been
previously detected by the classical analytical method.11–13
Furthermore, these new results led to a classification of the
toxin structures. These spider toxins consist of three struc-
tural elements: a lipophilic head; a polyamine backbone;
and a polyamine chain terminal, which are linearly con-
nected in this order. There are a variety of each element
O
H
N
H
N
putreanine
or Arg
R =
R
0-4
Ar
N
H
N
H
N
H
m
l
n
O
X
1-2
CONH2
X = O or H2; l, m, n = 1-2
polyamine backbone
lipophilic
head
polyamine chain terminal
Figure 1. Generalized structure of acylpolyamine spider toxins.
2. Results and discussion
2.1. Basic methodology
Keywords: Acylpolyamine; Spider toxins; Ns strategy; Structural classifica-
tion; Versatile synthesis.
* Corresponding author: Tel./fax: +55 11 3726 1024; e-mail: kk-gon@
butantan.gov.br
Present address: Department of Applied Biochemistry, Faculty of Agri-
y
Our synthetic strategy is based on the structural classification
asmentionedaboveandtheuseof2-nitrobenzenesulfonamide
culture, Utsunomiya University, Utsunomiya, Tochigi 321-0943, Japan
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.06.051

8336
K.-i. Nihei et al. / Tetrahedron 62 (2006) 8335–8350
OH
O
O
H
N
H
N
NH2
NH2
NH2
N
H
N
H
N
H
O
O
O
O
O
O
O
O
HO
CONH2 Type A: JSTX-3 (1)
O
O
H
N
H
N
N
H
N
H
N
H
N
H
CONH2 Type A: NPTX-8 (2)
OH
O
O
H
N
H
N
N
H
N
H
N
H
N
H
CONH2 Type A: NPTX-1 (3)
O
H
N
H
N
H
N
NH2
N
H
N
H
CONH2 Type B: NPTX-473 (4)
O
O
H
N
H
N
N
H
N
H
NH2
N
H
CONH2 Type C: NPTX-501 (5)
OH
O
O
NH2
H
N
H
N
H
N
NH2
NH
N
H
N
H
N
H
O
HO
HO
HO
CONH2 Type D: NSTX-3 (6)
O
O
H
N
N
H
N
H
N
H
NH2
O
CONH2 Type E: Joramine (7)
OH
O
NH
NH2
H
N
N
H
N
H
N
H
N
H
N
H
NH2
CONH2 Type F: Arg-636 (8)
Figure 2. Structures of acylpolyamine spider toxins.
(Ns or Nosyl) as an activating and/or protecting group (the Ns
strategy).18–20 The three structural elements can be consid-
ered as building blocks, that is, construction of the polyamine
backbone, followed by successive connection of the lipo-
philic head and polyamine chain terminal would afford a
variety of natural toxins as well as their analogs. Accordingly,
effective preparation of each polyamine backbone should
pave the way for convenient and versatile synthesis of these
toxins because this element has the richest variation among
those three elements (Fig. 2). In order to efficiently construct
the polyamine backbones, selective protection and/or activa-
tion of amino groups are needed. Fukuyama et al. reported
that using the Ns group as both protecting and activating
group is exceptionally versatile for the preparation of a vari-
ety of secondary amines (Ns strategies),18–20 and demon-
strated its utility for polyamine synthesis.21–23 Therefore,
their protocol is applied to the construction of the polyamine
backbones.
containing cadaverine as a diamine constituent. Thus, the
synthesis of 1 started from mono-Ns-cadaverine hydrochlo-
ride 9,21,22,25 which was readily coupled with commercially
available N-tBoc-L-asparagine-p-nitrophenyl ester for 0.5 h
to give the asparagyl cadaverine unit 10 (Scheme 1). Initial
attempts to remove the Ns group in 10 by the original proce-
dures of the Ns strategy led to poor results; for example,
treatment of 10 with thioacetic acid gave the corresponding
primary amine 11 only in 20% yield. These results prompted
us to improve the procedures for deprotection of 2-nitro-
benzenesulfonamide to corresponding primary amine, and
as a consequence, we were able to establish the modified
procedures using 2-mercaptoethanol/DBU or thiophenol/
Cs2CO3 in DMF.26 With these modified procedures, the pri-
mary amine 11 was obtained in high yield (>85%). On the
other hand, the carboxylic acid 13 was prepared from
mono-Ns-putrescine 1220,21 in excellent yield through
sequential reactions by the Michael addition to methyl acry-
late,27 protection by CbzCl, and hydrolysis of the methyl
ester. Coupling of the amine 11 with the succinimide ester
prepared from 13 by DCC afforded the polyamine backbone
precursor 14 in 90% yield. When acid anhydride method
with pivaloyl chloride or the coupling agent EDC for this
coupling reaction was used, the yields were less than 50%.
2.2. Synthesis of Type A toxins
JSTX-3 (1), isolated from the venom of Nephila clavata in
1986, is one of the first found acylpolyamine spider toxins.24
This polyamine backbone is classified into Type A,

K.-i. Nihei et al. / Tetrahedron 62 (2006) 8335–8350
8337
O
O
a
b
BocHN
BocHN
NHNs
NHNs
NHCbz
HCl H2N
NHNs
H2N
N
H
NHNs
N
H
NH2
CONH2
CONH2
9
10
11
c, d, e
NHNs
HO2C
N
Cbz
12
13
O
O
f, g
h
BocHN
O
N
H
N
H
N
Cbz
CONH2
14
O
Ns
N
i, j
BocHN
N
H
N
H
N
Cbz
CONH2
15
OBn
O
O
Ns
N
H
N
k, l
NHCbz
1
N
N
H
N
Cbz
H
O
BnO
CONH2
16
Scheme 1. Synthesis of JSTX-3 (1). Reagents and conditions: (a) Na-Boc-L-Asn-ONp, Et3N/DMF, 0 ꢀC to rt, 0.5 h, 97%; (b) 2-mercaptoethanol, DBU/DMF, rt,
2 h, 85%; (c) methyl acrylate/EtOH, rt, 5 h; (d) CbzCl, Et3N/CH2Cl2, 0 ꢀC to rt, 2 h, 86% (two steps); (e) NaOH/H2O–MeOH, 0 ꢀC to rt, 1 h, 96%; (f) HOSu,
DCC/CH2Cl2, 0 ꢀC, 5 h; (g) 11, DMF, rt, 0.5 h, 90% (two steps); (h) N-Cbz-3-bromopropylamine, Cs2CO3/DMF, 50 ꢀC, 1 h, 92%; (i) TFA/CHCl3, 0 ꢀC to rt,
1 h; (j) 2,4-dibenzyloxyphenylacetic acid-OSu, Et3N/DMF, rt, 2 h, 87% (two steps); (k) 2-mercaptoethanol, DBU/DMF, rt, 0.5 h; (l) H2-Pd(OH)2/AcOH, rt, 3 h,
66% (two steps).
Following the Ns strategy, the polyamine chain was elon-
gated by alkylation of 14 with N-Cbz-3-bromopropylamine
in the presence of Cs2CO3 to furnish the fully protected poly-
amine backbone 15 in 92% yield. After removal of the Boc
group in 15 by TFA, the resultant amine was condensed with
dibenzyl-2,4-dihydroxyphenylacetic acid N-hydroxysuc-
cinimide ester28 to give the fully protected JSTX-3 16 in
87% yield. Successive removal of the Ns and Cbz protective
groups by 2-mercaptoethanol and hydrogenation, respec-
tively, afforded 1 in high yield. Thus, JSTX-3 (1) was syn-
thesized from mono-Ns-cadaverine 9 in a 39% overall
yield via nine steps. The synthetic compound was identical
with authentic specimen in HPLC co-elution and compari-
son of the ESI-MS/MS spectra, and other spectroscopic
indoleacetate chromophore as the lipophilic head. There-
fore, coupling of the polyamine backbone 15 with appropri-
ate indoleacetate moieties instead of 2,4-dihydroxyphenyl
moiety would readily lead to these toxins. It was indeed
accomplished as shown in Scheme 2. The succinimide ester
of indoleacetic acid or 4-benzyloxyindoleacetic acid33 was
coupled with the amine derived from 15 by TFA gave the
fully protected toxins 17 or 18, respectively, from which
the natural toxins 2 and 3 were obtained by the same depro-
tection procedures as above. The synthetic compounds were
fully characterized by the spectroscopic data (IR, 1H and 13
C
NMR, FABHRMS, ESI-MS/MS), and identified with the
natural toxins.34,35 Thus, NPTX-8 and -1 were synthesized
from mono-Ns-cadaverine 9 in 35 and 36% overall yields,
respectively. In this way, a variety of Type A toxins and its
analogs can be synthesized.
1
data (IR, H and 13C NMR, FABHRMS) were consistent
with those previously reported.28
The synthesis of NPTX-8 (2) and -1 (3), originally isolated
from the Japanese spider N. clavata,29–31 and later found
in the Madagascarian spider Nephilengys borbonica11 and
Brazilian garden spider Nephilengys cruentata32 as well,
demonstrated the convenience of this synthetic method.
Both these toxins are also classified into Type A, containing
2.3. Synthesis of Type B toxin
The polyamine backbone of Type B has no amide bond in its
polyamine chain and the putrescine (C4) unit instead of the
cadaverine (C5) unit connects to the asparagine moiety. Ac-
cordingly, the alkylation procedures of the Ns strategy are
O
H
O
Ns
N
a, b
a, e
c, d
c, d
N
NHCbz
NHCbz
15
2
N
H
N
H
N
Cbz
O
O
N
H
CONH2
17
OBn
O
O
Ns
N
H
N
15
3
N
H
N
H
N
Cbz
N
H
CONH2
18
Scheme 2. Syntheses of NPTX-8 (2) and NPTX-1 (3). Reagents and conditions: (a) TFA/CHCl3, 0 ꢀC to rt, 1 h; (b) indoleacetic acid-OSu, Et3N/DMF, rt,
2 h, 84% (two steps); (c) 2-mercaptoethanol, DBU/DMF, rt, 0.5 h; (d) H2-Pd(OH)2/AcOH, rt, 2 h, 61% (two steps); (e) 4-benzyloxyindoleacetic acid-OSu,
Et3N/DMF, rt, 2 h, 86% (two steps).

8338
K.-i. Nihei et al. / Tetrahedron 62 (2006) 8335–8350
useful for construction of this polyamine chain, and the syn-
thesis should start with mono-Ns-putrescine 12.21,22 Scheme
3 shows the synthesis of Type B toxin NSTX-473 (4), found
in the venom gland of the Madagascarian spider N. borbon-
ica.11 Condensation of the putrescine unit 12 with N-tBoc-L-
asparagine-p-nitrophenyl ester furnished the acylputrescine
19 in 95% yield. On the other hand, mono-Ns-diaminopro-
pane 2021,22 was protected by CbzCl, and subsequently
alkylated with 1,3-dibromopropane to give the bromide 21
in high yield. Thus, the obtained two segments 19 and 21
were connected by the alkylation procedure of the Ns strat-
egy to afford the Type B polyamine backbone 22 in 94%
yield. Removal of the Boc protective group by TFA, fol-
lowed by coupling with indoleacetic acid succinimide ester
furnished the fully protected toxin 23 in 84% yield. Finally,
deprotection of the Ns and Cbz groups from 23 in the same
manner as for the Type A toxins afforded NPTX-473 (4) in
a 45% overall yield from 12 via six steps. The synthetic com-
pound was fully characterized by the spectroscopic data (IR,
1H and 13C NMR, FABHRMS, ESI-MS/MS), and consistent
with the proposed structure.11
yield the carboxylic acid 24 in high yield. Coupling the as-
paragine unit 11 with the succinimide ester prepared from
24 furnished the Type C acylpolyamine backbone 25 in ex-
cellent yield. Removal of the Boc group in 25 by TFA, fol-
lowed by coupling of the resultant amine with indoleacetic
acid succinimide ester afforded the fully protected toxin
26. Deprotection of the Ns and Cbz by the same procedures
as above produced NPTX-501 (5) in a 33% overall yield
from 12 via nine steps. The synthetic compound was fully
1
characterized by the spectroscopic data (IR, H and 13C
NMR, FABHRMS, ESI-MS/MS), and consistent with the
proposed structure.10,11
Ns
N
a, b, c
d, e
NHNs
HO2C
O
H2N
NHCbz
12
24
O
Ns
N
f, g
BocHN
N
N
NHCbz
H
H
CONH2
25
O
O
Ns
N
H
N
h, i
5
N
N
NHCbz
O
H
H
O
NHNs
BocHN
NHNs
a
N
H
CONH2
26
H2N
N
H
CONH2
12
19
Scheme 4. Synthesis of NPTX-501 (5). Reagents and conditions: (a) CbzCl,
Et3N/CH2Cl2, 0 ꢀC to rt, 1 h, 95%; (b) methyl bromoacetate, Cs2CO3/DMF,
50 ꢀC, 0.5 h, 86%; (c) NaOH/H2O–MeOH, 0 ꢀC to rt, 2 h, 86%; (d) HOSu,
DCC/CH2Cl2, 0 ꢀC, 4 h; (e) 11, DMF, rt, 0.5 h; (f) TFA/CHCl3, 0 ꢀC to rt,
1 h; (g) indoleacetic acid-OSu, Et3N/DMF, rt, 2 h, 73% (four steps); (h)
2-mercaptoethanol, DBU/DMF, rt, 0.5 h; (i) H2-Pd(OH)2/AcOH, rt, 2 h,
65% (two steps).
Ns
N
NsHN
NH2
Br
NHCbz
b, c
20
21
Ns
N
O
Ns
N
d
e, f
NHCbz
BocHN
N
H
2.5. Synthesis of Type D toxin
CONH2
22
Ns
N
O
Ns
N
H
The polyamine backbone of Type D is quite similar to that of
Type A and missing only the C3 unit at the right terminal.
Therefore, the toxins of these two types can be synthesized
from the common polyamine precursor 14. Thus, the Type
D toxin NSTX-3 (6), isolated from the Papua New Guinean
spider Nephila maculata,24 was synthesized in a manner
similar to that of Type A toxins (Schemes 1 and 2) as shown
in Scheme 5. Removal of the Ns protective group of 14,26
followed by condensation with tri-Cbz-arginine succinimide
ester furnished 27 in 76% yield. Deprotection of the Boc pro-
tective group of 28 by TFA, and subsequent condensation
with 2,4-dihydroxyphenylacetic acid N-hydroxysuccinimide
ester afforded the fully protected toxin 14 in 87% yield.
Finally, hydrogenation of 28 afforded NSTX-3 (6) in a
36% overall yield from 9 via nine steps. The synthetic com-
pound was fully characterized by the spectroscopic data (IR,
1H and 13C NMR, FABHRMS, ESI-MS/MS), and identified
with the natural24 and synthetic toxins.39–41
g, h
NHCbz
N
4
N
H
O
N
H
CONH2
23
Scheme 3. Synthesis of NPTX-473 (4). Reagents and conditions: (a) Na-
Boc-L-Asn-ONp, DMF, 0 ꢀC to rt, 2 h, 95%; (b) CbzCl, Et3N/CH2Cl2,
0 ꢀC to rt, 2 h, 88%; (c) 1,3-dibromopropane, Cs2CO3/DMF, 50 ꢀC, 0.5 h,
91%; (d) 19, TBAI, Cs2CO3/DMF, 70 ꢀC, 1 h, 94%; (e) TFA/CHCl3, 0 ꢀC
to rt, 1 h; (f) indoleacetic acid-OSu, Et3N/DMF, rt, 2 h, 84% (two steps);
(g) 2-mercaptoethanol, DBU/DMF, rt, 0.5 h; (h) H2-Pd(OH)2/AcOH, rt,
2 h, 60% (two steps).
It is noteworthy that the structure of this polyamine chain is
the same as that of PhTX-433, a glutamate receptor antago-
nist from the venom of the solitary wasp Philanthus triangu-
lum.36,37 Therefore, the method established here is
applicable to the synthesis of PhTX-433 and its deriva-
tives.38
2.4. Synthesis of Type C toxin
2.6. Synthesis of Type E toxin
The Type C structure is close to but simpler than that of Type
A; that is, the b-alanine moiety is replaced by the glycine
moiety and the C3 unit of the right terminal is missing.
Scheme 4 shows the synthesis of NPTX-501 (5), a Type C
toxin found in Madagascarian spiders Nephilengys mada-
gascariensis and N. borbonica.10,11 The primary amine of
mono-Ns-putrescine 12 was protected by CbzCl, then alky-
lated with methyl bromoacetate by the Ns strategy at 50 ꢀC
for 0.5 h and the resultant methyl ester was hydrolyzed to
The Type E polyamine backbone is only slightly different
from Type D, that is, the C3 unit is on the right terminal
instead of the C4 unit. Accordingly, starting with mono-
Ns-1,3-diaminopropane 20 and using the same procedures
as that for the Type D would give Type E toxin. Scheme 6
shows the synthesis of joramine (7), a minor component of
the venom of N. clavata.42 The carboxylic acid 29 was
obtained from a series of reactions with Michael addition,

K.-i. Nihei et al. / Tetrahedron 62 (2006) 8335–8350
8339
O
O
NHCbz
Cbz
N
H
N
a, b
c, d
NHCbz
BocHN
14
N
H
N
H
N
Cbz
O
NH
CONH2
27
OBn
O
O
NHCbz
Cbz
N
H
N
H
N
e
NHCbz
N
H
N
H
N
Cbz
6
O
O
NH
BnO
CONH2
28
Scheme 5. Synthesis of NSTX-3 (6). Reagents and conditions: (a) 2-mercaptoethanol, DBU/DMF, rt, 0.5 h; (b) tri-Cbz-arginine-OSu, DMF, rt, 2 h; 76% (two
steps); (c) TFA/CHCl3, 0 ꢀC to rt, 1 h; (d) 2,4-dibenzyloxyphenylacetic acid-OSu, Et3N/DMF, rt, 2 h, 87% (two steps); (e) H2-Pd(OH)2/AcOH, rt, 4 h, 74%.
Cbz protection, and ester hydrolysis. The acid 29 was cou-
pled with the amine unit 11 to afford the polyamine back-
bone 30 in 82% yield. After removal of the Boc group by
TFA, the resultant amine was coupled with 4-benzyloxy-
phenylacetic acid succinimide ester to furnish the fully
protected toxin 31, which was converted to joramine (7)
by the usual manner. Thus, the Type E toxin joramine (7)
was synthesized in a 36% overall yield from 20 via nine
steps. The synthetic compound was fully characterized by
2.7. Synthesis of Type F toxin
The Type F polyamine structure is quite similar to that of
Type B; the only difference is the cadaverine (C5) unit
instead of the putrescine (C4) unit connected to asparagine.
Accordingly, this polyamine backbone can be constructed
by basically the same way as used for Type B. Argiotoxin-
636 (Argiopine, 8), isolated from the Argiope spiders43,44
in 1986 as well as JSTX-3,45 can be classified into Type F
and its synthesis is shown in Scheme 7. In this case, however,
in order to attach arginine moiety at the polyamine terminal,
selective protection of the terminal primary amine of the
polyamine backbone is necessary. Thus, allyloxycarbonyl
(Alloc) group was used for the third protective group for
construction of polyamine chain. Mono-Ns-diaminopropane
20 was converted to the corresponding allyl carbamate,
which was further alkylated by 1,3-dibromopropane in the
presence of Cs2CO3 at 50 ꢀC. By the same way as used for
Type B toxin, the resultant bromide 32 was coupled with
the asparagine/cadaverne conjugate 10 to afford the Type
F polyamine backbone 33 in excellent yield. The Alloc moi-
ety in 33 was selectively removed using tetrakis-(triphenyl-
phosphine)palladium(0) (Pd(PPh3)4) as a catalyst and
pyrrolidine as a nucleophile at room temperature in high
yield.46 Instead of Pd(PPh3)4, tris(dibenzylideneacetone)-
dipalladium(0) was also effective for this deprotection.
Thus, the obtained primary amine was coupled with the
arginine derivative by its N-hydroxysuccinimide ester to
give 34. Boc removal and subsequent acylation of 34,
followed by deprotection in the usual manner afforded
1
the spectroscopic data (IR, H and 13C NMR, FABHRMS,
ESI-MS/MS), and identified with the natural toxin.42
a, b, c
d, e
f, g
HO2C
H2N
NHNs
O
N
Cbz
NHNs
NHNs
20
29
O
BocHN
N
H
N
H
N
Cbz
CONH2
30
O
O
H
N
h, i
7
N
H
N
H
N
Cbz
NHNs
O
BnO
CONH2
31
Scheme 6. Synthesis of joramine (7). Reagents and conditions: (a) methyl
acrylate/EtOH, rt, 5 h; (b) CbzCl, Et3N/CH2Cl2, 0 ꢀC to rt, 2 h, 86% (two
steps); (c) NaOH/H2O–MeOH, 0 ꢀC to rt, 1 h, 85%; (d) HOSu, DCC/
CH2Cl2, 0 ꢀC, 5 h; (e) 11, DMF, rt, 0.5 h, 82% (two steps); (f) TFA/
CHCl3, 0 ꢀC to rt, 1 h; (g) 4-benzyloxyphenylacetic acid-OSu, Et3N/
DMF, rt, 2 h, 88% (two steps); (h) 2-mercaptoethanol, DBU/DMF, rt,
0.5 h; (i) H2-Pd(OH)2/AcOH, rt, 3 h, 69% (two steps).
a, b
c
NsHN
NH2
Br
N
Ns
NHAlloc
NHAlloc
20
32
O
d, e
BocHN
N
H
N
Ns
N
Ns
CONH2
33
O
O
NH
NHCbz
f, g
BocHN
OBn
N
H
N
Ns
N
Ns
N
H
N
Cbz
NHCbz
O
CONH2
34
O
NH
NHCbz
H
N
h, i
8
N
H
N
Ns
N
Ns
N
H
N
O
NHCbz
Cbz
BnO
CONH2
35
Scheme 7. Synthesis of argiotoxin-636 (8). Reagents and conditions: (a) AllocCl, Et3N/CH2Cl2, 0 ꢀC, 0.5 h, 93%; (b) 1,3-dibromopropane, Cs2CO3/DMF,
50 ꢀC, 0.5 h, 90%; (c) 10, TBAI, Cs2CO3/DMF, 70 ꢀC, 1 h; (d) Pd(PPh3)4, PPh3, pyrrolidine/CH2Cl2, rt, 0.5 h; (e) tri-Cbz-arginine-OSu, DMF, rt, 1 h, 66%
(three steps); (f) TFA/CHCl3, 0 ꢀC to rt, 1 h; (g) 2,4-dibenzyloxyphenylacetic acid-OSu, Et3N/DMF, rt, 2 h, 80% (two steps); (h) 2-mercaptoethanol, DBU/
DMF, rt, 0.5 h; (i) H2-Pd(OH)2/AcOH, rt, 2 h, 51% (two steps).

8340
K.-i. Nihei et al. / Tetrahedron 62 (2006) 8335–8350
argiotoxin-636 (8) in a 26% overall yield from mono-Ns
cadaverine 9 via eight steps. The synthetic compound was
fully characterized by the spectroscopic data (IR, H and
J¼6.6 Hz), 4.29 (dd, 1H, J¼6.0, 6.3 Hz), 7.75 (m, 3H),
8.00 (m, 1H). 13C NMR (75 MHz, CD3OD): d 24.7, 28.7,
29.7, 30.4, 38.3, 40.2, 44.2, 53.0, 80.9, 125.8, 131.5,
133.5, 134.9, 149.6, 157.5, 173.8, 175.1. HRMS calcd for
C14H27N4O4 (M+ꢂNs) 315.2032, found 315.2036.
1
13C NMR, FABHRMS, ESI-MS/MS), and identified with
the natural43,44 and synthetic toxins.47–50
4.1.2. N-[Na-(tert-Butoxycarbonyl)-L-asparaginyl]-1,5-
diaminopentane (11). To a stirred solution of sulfonamide
10 (500 mg, 997 mmol) in DMF (2 mL) were added 2-mer-
captoethanol (234 mg, 3.00 mmol) and DBU (456 mg,
3.00 mmol) slowly. After being stirred for 0.5 h at room tem-
perature, the reaction mixture was directly subjected to silica
gel chromatography (1–8% MeOH/CH2Cl2, then, 7.5%
MeOH/CHCl3 containing 2.5% i-PrNH2) to afford the title
compound 11 (269 mg, 85%) as a white powder. Spectral
data (1H and 13C NMR, IR, MS) obtained were completely
in agreement with those reported previously.53
3. Conclusion
We have established an efficient and versatile synthesis of
acylpolyamine spider toxins based on the structural classifi-
cation of the Nephila and Nephilengys spider toxins using the
2-nitrobenzenesulfonamide group (the Ns strategy). The nat-
urally occurring toxins 1–8 corresponding to each structural
type have been efficiently synthesized by this method in
a high overall yield with few steps. This method is soversatile
that it would enable us to synthesize a variety of analogues as
well as naturally occurring toxins. Therefore, it would be
highly useful for structure–activity relationship and mode
of action studies of the acylpolyamine toxins in more detail.
Studies along this line are currently underway.51,52
4.1.3. Methyl 8-(2-nitrobenzenesulfonylamino)-4-benzyl-
oxycarbonyl-4-azaoctanoate. A solution of methyl acrylate
(158 mg, 1.84 mmol) in EtOH (10 mL) was added at room
temperature over 5 h to a stirring solution of N-(5-amino-
butyl)-2-nitrobenzenesulfonamide22 (12) (500 mg, 1.83 mmol)
in EtOH (20 mL). The solvent was evaporated in vacuo,
and the residue was subjected to silica gel chromatography
(5–20% MeOH/CH2Cl2) to give the secondary amine as a
colorless oil, which was used in the next step without further
purification.
4. Experimental
4.1. General
Optical rotation was measured on a Jasco DIP-370 polari-
meter. IR spectra were recorded on a Perkin–Elmer infrared
spectrometer model 1750. NMR spectra were measured on
a Varian DPX-300 spectrometer. Low and high resolution
mass spectra were recorded on a JEOL JMS D-300 mass
spectrometer. ESI-MS/MS spectra were measured on a
Micromass Q-TOF Ultima API fitted with an electrospray
ion source in positive ionization mode. Preparative HPLC
was performed on a Shimadzu LC-10 instrument equipped
with a CAPCELL PAK C-18 (5 mm, 10ꢁ250 mm) with
a flow rate of 5 mL minꢂ1 and detection at 210 nm. Column
chromatography was carried out on silica gel 60 (70–230
To a cold (0 ꢀC) and stirred solution of the secondary amine
in CH2Cl2 (20 mL) were added CbzCl (30% in toluene,
1.04 g, 1.83 mmol) and Et3N (185 mg, 1.83 mmol) slowly.
After being stirred for 2 h at room temperature, the reaction
mixture was diluted with EtOAc (200 mL) and washed with
saturated NH4Cl solution (3ꢁ50 mL) and brine (3ꢁ50 mL).
Aqueous layers were extracted with EtOAc (3ꢁ40 mL) and
combined organic layers were dried over Na2SO4. Filtration
and concentration followed by chromatography on silica gel
(30–50% EtOAc/hexane) gave methyl ester (775 mg, 86%,
two steps) as a colorless oil. IR (neat) 1735, 1696, 1542,
mesh), and preparative TLC was run on silica gel 60F254
.
All dried solvents were purchased from Aldrich Chemical
Co. The authentic specimen of JSTX-3 was purchased
from Wako Chemical Co.
1422, 1365, 1166, 853 cmꢂ1 1H NMR (300 MHz,
.
CDCl3): d 1.54 (m, 4H), 2.57 (m, 2H), 3.09 (m, 2H), 3.25
(t, 2H, J¼6.3 Hz), 3.49 (t, 2H, J¼7.2 Hz), 3.65 (s, 3H),
5.11 (s, 2H), 5.48 (br s, 1H), 7.34 (m, 5H), 7.73 (m, 2H),
7.84 (m, 1H), 8.11 (m, 1H). 13C NMR (75 MHz, CDCl3):
d 25.0, 26.7, 33.7, 43.0, 43.3, 47.2, 51.8, 67.2, 125.4,
127.9, 128.1, 128.5, 131.0, 132.7, 133.5, 133.7, 136.5,
148.0, 155.9, 172.0. HRMS calcd for C21H24N3O7S
(M+ꢂOMe) 462.1335, found 462.1353.
4.1.1. N-[Na-(tert-Butoxycarbonyl)-L-asparaginyl]-N0-
(2-nitrobenzenesulfonyl)-1,5-diaminopentane (10). To a
stirred solution of N-(5-amino-pentyl)-2,4-dinitro-benzene-
sulfonamide HCl salt22 (9) (500 mg, 1.54 mmol) in DMF
(5 mL) were added Na-Boc-L-asparagine-p-nitrophenyl
ester (544 mg, 1.54 mmol) and Et3N (156 mg, 1.54 mmol)
at 0 ꢀC. After being stirred for 0.5 h at room temperature,
the reaction mixture was diluted with EtOAc (200 mL)
and washed with saturated aqueous NaHCO3 solution
(5ꢁ50 mL) and brine (3ꢁ50 mL). Aqueous layers were
extracted with EtOAc (3ꢁ30 mL) and combined organic
layers were dried over Na2SO4. Filtration and concentration
followed by chromatography on silica gel (1–4% MeOH/
CH2Cl2) gave the title compound 10 (745 mg, 97%) as
a white powder. [a]2D4 ꢂ3.85 (c 2.83, MeOH). IR (Nujol)
4.1.4. 8-(2-Nitrobenzenesulfonylamino)-4-benzyloxycar-
bonyl-4-azaoctanoic acid (13). To a cold (0 ꢀC) and stirred
solution of the above methyl ester (700 mg, 1.42 mmol) in
MeOH (5 mL) was added 3.0 M aqueous NaOH solution
(1.42 mL, 4.26 mmol) slowly. After being stirred for 1 h at
room temperature, the reaction mixture was adjusted to pH
2.0 by concentrated HCl. The resultant mixture was diluted
with EtOAc (100 mL) and washed with H2O (3ꢁ20 mL) and
brine (3ꢁ20 mL). Aqueous layers were extracted with
EtOAc (3ꢁ20 mL) and combined organic layers were dried
over Na2SO4. Filtration and concentration followed by chro-
matography on silica gel (50% EtOAc/hexane, then, EtOAc)
gave the title compound 13 (652 mg, 96%) as a colorless oil.
1683, 1657, 1542, 1461, 1162, 856 cmꢂ1 1H NMR
.
(300 MHz, CD3OD): d 1.26 (m, 2H), 1.36 (s, 9H), 1.41
(m, 4H), 2.52 (dd, 1H, J¼6.3, 15.3 Hz), 2.58 (dd, 1H,
J¼6.0, 15.3 Hz), 2.96 (t, 2H, J¼6.9 Hz), 3.07 (t, 1H,

K.-i. Nihei et al. / Tetrahedron 62 (2006) 8335–8350
8341
IR (film) 1686, 1541, 1424, 1365, 1165, 854 cmꢂ1. 1H NMR
(300 MHz, CDCl3): d 1.54 (m, 4H), 2.61 (m, 2H), 3.10 (m,
2H), 3.26 (t, 2H, J¼6.0 Hz), 3.50 (t, 2H, J¼7.2 Hz), 5.11
(s, 2H), 5.54 (br s, 1H), 7.36 (m, 5H), 7.71 (m, 2H), 7.82
(m, 1H), 8.10 (m, 1H). 13C NMR (75 MHz, CDCl3):
d 25.0, 26.7, 33.0, 42.9, 43.3, 47.5, 67.3, 125.3, 127.9,
128.1, 128.6, 131.0, 132.8, 133.6, 133.7, 136.4, 148.0,
156.1, 176.3. HRMS calcd for C13H18N3O6S (M+ꢂCbz)
344.0917, found 344.0917.
(m, 4H), 3.16 (t, 2H, J¼6.3 Hz), 3.27 (m, 6H), 3.48 (m,
2H), 4.36 (dd, 1H, J¼5.7, 6.0 Hz), 5.05 (s, 2H), 5.09 (s,
2H), 7.34 (m, 10H), 7.73 (m, 3H), 7.96 (m, 1H). 13C NMR
(75 MHz, CD3OD): d 25.1, 26.4, 28.7, 29.8, 29.9, 30.0,
36.6, 38.4, 39.1, 40.3, 45.6, 46.4, 53.1, 67.4, 68.3, 80.9,
125.4, 128.8, 129.0, 129.2, 129.5, 129.6, 131.4, 133.1,
134.0, 135.2, 138.1, 138.4, 149.6, 157.8, 158.8, 173.6,
173.8, 175.1. FABHRMS calcd for C46H64N8O13SNa
(M+Na)+ 991.4211, found 991.4217.
4.1.5. N-[Na-(tert-Butoxycarbonyl)-L-asparaginyl]-N0-[8-
(2-nitrobenzenesulfonylamino)-4-benzyloxycarbonyl-4-
azaoctanoyl]-1,5-diaminopentane (14). To a cold (0 ꢀC)
and stirred suspension of carboxylic acid 13 (363 mg,
757 mmol) and HOSu (175 mg, 1.52 mmol) in CH2Cl2
(5 mL) was added DCC (235 mg, 1.14 mmol) slowly. After
being stirred for 5 h at 0 ꢀC, the reaction mixture was fil-
trated through a pad of silica gel with EtOAc. The filtrate
was concentrated in vacuo to give the crude succinimide
ester as a colorless oil.
4.1.7. N-[Na-(2,4-Dibenzyloxyphenylacetyl)-L-aspara-
ginyl]-N0-[12-benzyloxycarbonylamino-9-(2-nitrobenz-
enesulfonyl)-4-benzyloxycarbonyl-4,9-diazaundecanoyl]-
1,5-diaminopentane (16). To a cold (0 ꢀC) and stirred sus-
pension of polyamine 15 (165 mg, 170 mmol) in CHCl3
(1 mL) was added TFA (1 mL) slowly. After being stirred
for 1 h at room temperature, the reaction mixture was
concentrated in vacuo to give the crude amine TFA salt as
a colorless oil.
To a stirred solution of the amine TFA salt in DMF (1 mL)
were added Et3N (18 mg, 178 mmol) and 2,4-dibenzyloxy-
phenylacetic acid succinimidyl ester28 (116 mg, 260 mmol)
slowly. After being stirred for 2 h at room temperature, the
reaction mixture was directly subjected to silica gel chroma-
tography (1–10% EtOH/CH2Cl2) to afford the title com-
pound 16 (177 mg, 87%, two steps) as a white powder.
[a]2D2 ꢂ0.75 (c 2.01, DMF). IR (Nujol) 1695, 1664, 1642,
1544, 1463, 1173, 852 cmꢂ1. 1H NMR (300 MHz, DMSO-
d6): d 1.16 (quin, 2H, J¼6.9 Hz), 1.29 (m, 4H), 1.38 (m,
4H), 1.59 (m, 2H), 2.28 (t, 2H, J¼6.6 Hz), 2.35 (dd, 1H,
J¼7.5, 15.3 Hz), 2.46 (dd, 1H, J¼6.3, 15.3 Hz), 2.95 (m,
6H), 3.21 (m, 8H), 3.41 (s, 2H), 4.50 (dd, 1H, J¼6.6,
7.5 Hz), 4.99 (s, 2H), 5.04 (s, 4H), 5.08 (s, 2H), 6.52 (d,
1H, J¼8.4 Hz), 6.68 (s, 1H), 6.85 (s, 1H), 7.07 (d, 1H,
J¼8.1 Hz), 7.32 (m, 22H), 7.60 (t, 1H, J¼5.4 Hz), 7.82
(m, 3H), 7.95 (m, 1H). 13C NMR (75 MHz, CD3OD):
d 23.8, 25.3, 28.6, 28.8, 28.9, 34.4, 35.1, 36.4, 37.5, 38.0,
38.5, 38.7, 44.0, 45.5, 46.5, 47.4, 50.0, 65.4, 66.2, 69.4,
69.5, 100.7, 105.9, 117.3, 124.4, 127.3, 127.7, 127.9,
128.0, 128.5, 128.6, 129.8, 131.1, 132.0, 132.6, 134.6,
137.3, 137.4, 147.8, 155.3, 156.3, 157.1, 158.5, 170.1,
170.5, 170.9, 171.8. FABHRMS calcd for C63H74N8O14SNa
(M+Na)+ 1221.4943, found 1221.4943.
To a stirred solution of obtained ester in DMF (6 mL) was
added amine 11 (241 mg, 762 mmol) slowly. After being
stirred for 0.5 h at room temperature, the reaction mixture
was diluted with EtOAc (100 mL) and washed with satu-
rated aqueous NaHCO3 solution (3ꢁ20 mL) and brine
(3ꢁ20 mL). Aqueous layers were extracted with EtOAc
(3ꢁ20 mL) and combined organic layers were dried over
Na2SO4. Filtration and concentration followed by chromato-
graphy on silica gel (2–8% EtOH/CH2Cl2) gave the title
compound 14 (532 mg, 90%, two steps) as a colorless solid.
[a]2D4 ꢂ2.61 (c 2.30, MeOH). IR (Nujol) 1674, 1542, 1449,
1369, 1165, 854 cmꢂ1 1H NMR (300 MHz, CD3OD):
.
d 1.21 (m, 2H), 1.32 (s, 9H), 1.36 (m, 8H), 2.30 (t, 2H,
J¼6.6 Hz), 2.47 (dd, 1H, J¼6.6, 12.3 Hz), 2.53 (dd, 1H,
J¼5.7, 12.3 Hz), 2.92 (m, 2H), 3.02 (t, 2H, J¼7.2 Hz),
3.06 (t, 2H, J¼6.9 Hz), 3.13 (t, 2H, J¼6.9 Hz), 3.37 (m,
2H), 4.26 (dd, 1H, J¼5.7, 6.6 Hz), 4.98 (s, 2H), 7.23 (m,
5H), 7.68 (m, 3H), 7.93 (m, 1H). 13C NMR (75 MHz,
CD3OD): d 25.1, 26.6, 28.0, 28.7, 29.8, 29.9, 36.5, 38.4,
40.3, 40.5, 44.0, 45.6, 53.1, 68.3, 80.9, 125.8, 128.9,
129.1, 129.6, 131.5, 133.5, 134.9, 135.0, 138.1, 149.6,
157.5, 157.8, 173.6, 173.8, 175.1. FABHRMS calcd for
C35H51N7O11SNa (M+Na)+ 800.3265, found 800.3276.
4.1.6. N-[Na-(tert-Butoxycarbonyl)-L-asparaginyl]-N0-
[12-benzyloxycarbonylamino-9-(2-nitrobenzenesulfonyl)-
4-benzyloxycarbonyl-4,9-diazaundecanoyl]-1,5-diami-
nopentane (15). To a solution of sulfonamide 14 (200 mg,
257 mmol) and N-Cbz-3-bromopropylamine53 (106 mg,
390 mmol) in DMF (2 mL) was added Cs2CO3 (169 mg,
519 mmol). After being stirred for 1 h at 50 ꢀC, the reaction
mixture was diluted with EtOAc (100 mL) and washed with
H2O (3ꢁ20 mL) and brine (3ꢁ20 mL). Aqueous layers were
extracted with EtOAc (3ꢁ20 mL) and combined organic
layers were dried over Na2SO4. Filtration and concentration
followed by chromatography on silica gel (2–8% MeOH/
CH2Cl2) gave the title compound 15 (230 mg, 92%) as a col-
orless oil. [a]2D2 ꢂ2.28 (c 2.46, MeOH). IR (Nujol) 1688,
4.1.8. JSTX-3 (1). To a stirred solution of polyamine 16
(40 mg, 33.4 mmol) in DMF (1 mL) were added 2-mer-
captoethanol (8.0 mg, 102 mmol) and DBU (15 mg,
98.5 mmol). After being stirred for 0.5 h at room tempera-
ture, the reaction mixture was directly purified on silica
gel column (2–12% EtOH/CH2Cl2, then, 7.5% MeOH/
CHCl3 containing 2.5% i-PrNH2) and then, further purifica-
tion by preparative TLC (7.5% MeOH/CHCl3 containing
2.5% i-PrNH2) gave the secondary amine as a white powder.
A solution of the secondary amine in AcOH (1 mL) was hy-
drogenated over 20% Pd(OH)2 on carbon (20 mg) for 3 h.
The mixture was filtrated through Celite and the filtrate
was evaporated to dryness. The resultant residue was dis-
solved in water and was purified by preparative RP-HPLC
with a linear gradient from 5% H2O/MeCN containing
0.1% TFA to 50% H2O/MeCN containing 0.1% TFA in
20 min. JSTX-3 (1) was eluted at 9.54 min and was obtained
1667, 1636, 1546, 1462, 1163, 852 cmꢂ1 1H NMR
.
(300 MHz, CD3OD): d 1.30 (m, 2H), 1.43 (s, 9H), 1.48
(m, 8H), 1.69 (m, 2H), 2.41 (t, 2H, J¼6.0 Hz), 2.56 (dd,
1H, J¼6.0, 16.5 Hz), 2.62 (dd, 1H, J¼5.7, 16.5 Hz), 3.10

8342
K.-i. Nihei et al. / Tetrahedron 62 (2006) 8335–8350
as a colorless TFA salt (20 mg, 66%, two steps). [a]2D2 ꢂ4.74
(c 0.97, H2O). IR (Nujol) 3518, 2924, 1695, 1547 cmꢂ1. 1H
NMR (300 MHz, D2O): d 1.04 (quin, 2H, J¼7.8 Hz), 1.29
(m, 4H), 1.63 (m, 4H), 1.94 (quin, 2H, J¼7.5 Hz), 2.50 (t,
2H, J¼6.9 Hz), 2.60 (dd, 1H, J¼7.5, 15.3 Hz), 2.64 (dd,
1H, J¼5.7, 15.3 Hz), 2.96 (m, 12H), 3.14 (t, 2H,
J¼6.9 Hz), 3.34 (d, 1H, J¼15.3 Hz), 3.44 (d, 1H,
J¼15.3 Hz), 4.46 (dd, 1H, J¼5.7, 7.5 Hz), 6.31 (m, 2H),
6.95 (d, 1H, J¼9.0 Hz). 13C NMR (75 MHz, D2O): d 22.5,
22.6, 23.1, 23.7, 27.6, 27.7, 30.8, 35.9, 36.4, 36.8, 39.1,
39.2, 43.4, 44.4, 46.7, 46.9, 50.8, 102.8, 107.4, 113.7,
132.4, 155.1, 156.1, 171.5, 172.3, 174.6, 174.9. FABHRMS
calcd for C27H48N7O6 (M+H)+ 566.3670, found 566.3660.
ESI-MS/MS: Supplementary data 1 and 2.
A solution of the secondary amine in AcOH (1 mL) was
hydrogenated over 20% Pd(OH)2 on carbon (20 mg) for
2 h. The mixture was filtrated through Celite and the filtrate
was evaporated to dryness. The resultant residue was
dissolved in water and was purified by preparative RP-
HPLC with a linear gradient from 5% H2O/MeCN contain-
ing 0.1% TFA to 50% H2O/MeCN containing 0.1% TFA in
20 min. NPTX-8 (2) was eluted at 12.26 min and was
obtained as a colorless TFA salt (22 mg, 61%, two steps).
[a]2D2 ꢂ2.86 (c 0.77, H2O). IR (Nujol) 3481, 2924, 1697,
1
1539 cmꢂ1. H NMR (300 MHz, D2O): d 1.00 (quin, 2H,
J¼7.2 Hz), 1.23 (quin, 4H, J¼7.2 Hz), 1.60 (m, 4H),
1.94 (quin, 2H, J¼8.4 Hz), 2.46 (t, 2H, J¼6.6 Hz), 2.52
(dd, 1H, J¼7.5, 15.3 Hz), 2.61 (dd, 1H, J¼5.7, 15.3 Hz),
2.94 (m, 12H), 3.09 (t, 2H, J¼6.6 Hz), 3.67 (s, 2H), 4.49
(dd, 1H, J¼5.7, 7.5 Hz), 7.04 (t, 1H, J¼7.8 Hz), 7.13 (t,
1H, J¼7.8 Hz), 7.20 (s, 1H), 7.39 (d, 1H, J¼7.8 Hz),
7.46 (d, 1H, J¼7.8 Hz). 13C NMR (75 MHz, D2O):
d 22.5, 22.6, 23.0, 23.6, 27.5, 27.6, 30.7, 32.2, 36.1,
36.4, 39.0, 39.1, 43.4, 44.4, 46.6, 46.9, 50.8, 107.4,
111.9, 118.2, 119.5, 122.1, 124.9, 126.5, 136.2, 171.4,
172.1, 174.4, 175.0. FABHRMS calcd for C29H49N8O4
(M+H)+ 573.3895, found 573.3853. ESI-MS/MS: Supple-
mentary data 3.
4.1.9. N-[Na-(Indoleacetyl)-L-asparaginyl]-N0-[12-benzyl-
oxycarbonylamino-9-(2-nitrobenzenesulfonyl)-4-benzyl-
oxycarbonyl-4,9-diazaundecanoyl]-1,5-diaminopentane
(17). To a cold (0 ꢀC) and stirred suspension of polyamine
15 (115 mg, 119 mmol) in CHCl3 (1 mL) was added TFA
(1 mL) slowly. After being stirred for 1 h at room tempera-
ture, the reaction mixture was concentrated in vacuo to
give the crude amine TFA salt as a colorless solid.
To a stirred solution of obtained amine TFA salt in DMF
(1 mL) were added TEA (13 mg, 129 mmol) and indole-
acetic acid succinimide ester45 (49 mg, 180 mmol) slowly.
After being stirred for 2 h at room temperature, the reaction
mixture was diluted with EtOAc (100 mL) and washed
with saturated NaHCO3 solution (3ꢁ20 mL) and brine
(3ꢁ20 mL). Aqueous layers were extracted with EtOAc
(3ꢁ20 mL) and combined organic layers were dried over
Na2SO4. Filtration and concentration followed by chroma-
tography on silica gel (2–10% EtOH/CH2Cl2) gave the title
compound 17 (102 mg, 84%, two steps) as a white powder.
[a]2D4 +2.55 (c 0.51, MeOH). IR (Nujol) 1668, 1543, 1461,
4.1.11. N-[Na-(4-Benzyloxyindoleacetyl)-L-asparaginyl]-
N0-[12-benzyloxycarbonylamino-9-(2-nitrobenzenesul-
fonyl)-4-benzyloxycarbonyl-4,9-diazaundecanoyl]-1,5-
diaminopentane (18). To a cold (0 ꢀC) and stirred suspen-
sion of polyamine 15 (200 mg, 206 mmol) in CHCl3
(1 mL) was added TFA (1 mL) slowly. After being stirred
for 1 h at room temperature, the reaction mixture was
concentrated in vacuo to give the crude amine TFA salt as
a colorless solid.
To a stirred solution of obtained amine TFA salt in DMF
(1 mL) were added TEA (21 mg, 208 mmol) and 4-benzyl-
oxyindoleacetic acid succinimide ester33 (117 mg,
309 mmol) slowly. After being stirred for 2 h at room tem-
perature, the reaction mixture was diluted with EtOAc
(100 mL) and washed with saturated NaHCO3 solution
(3ꢁ20 mL) and brine (3ꢁ20 mL). Aqueous layers were ex-
tracted with EtOAc (3ꢁ20 mL) and combined organic
layers were dried over Na2SO4. Filtration and concentration
followed by chromatography on silica gel (2–10% EtOH/
CH2Cl2) gave the title compound 18 (201 mg, 86%, two
steps) as a white powder. [a]2D2 +8.07 (c 1.50, DMF). IR
1
1250, 1161, 852 cmꢂ1. H NMR (300 MHz, DMSO-d6):
d 1.17 (m, 2H), 1.28 (m, 4H), 1.38 (m, 4H), 1.60 (m,
2H), 2.29 (t, 2H, J¼6.6 Hz), 2.36 (dd, 1H, J¼6.9,
15.3 Hz), 2.48 (dd, 1H, J¼6.0, 15.3 Hz), 2.95 (m, 6H),
3.22 (m, 8H), 3.54 (s, 2H), 4.50 (dd, 1H, J¼6.0, 6.9 Hz),
4.99 (s, 2H), 5.03 (s, 2H), 6.84 (s, 1H), 6.94 (t, 1H,
J¼7.8 Hz), 7.04 (t, 1H, J¼7.8 Hz), 7.18 (s, 1H), 7.32 (m,
12H), 7.52 (d, 1H, J¼7.5 Hz), 7.58 (d, 1H, J¼5.1 Hz),
7.82 (m, 3H), 7.95 (m, 2H), 8.09 (d, 1H, J¼8.1 Hz),
10.85 (s, 1H). 13C NMR (75 MHz, CD3OD): d 23.8, 25.3,
28.7, 28.8, 28.9, 32.7, 35.1, 37.5, 38.0, 38.6, 38.7, 43.2,
44.1, 45.5, 46.5, 47.4, 50.0, 65.4, 66.2, 108.9, 111.5,
118.5, 118.8, 121.1, 124.0, 124.4, 127.4, 127.9, 128.0,
128.5, 128.6, 129.8, 132.0, 132.6, 134.6, 136.3, 137.2,
137.4, 147.7, 155.4, 156.3, 170.2, 170.8, 170.9, 171.7.
FABHRMS calcd for C51H63N9O12SNa (M+Na)+
1048.4214, found 1048.4216.
(Nujol) 1698, 1665, 1639, 1542, 1376, 1248 cmꢂ1 1H
.
NMR (300 MHz, DMSO-d6): d 1.16 (m, 2H), 1.28 (m,
4H), 1.39 (m, 4H), 1.61 (m, 2H), 2.29 (t, 2H, J¼6.6 Hz),
2.38 (m, 2H), 2.95 (m, 6H), 3.22 (m, 8H), 3.74 (s, 2H),
4.51 (m, 1H), 4.99 (s, 2H), 5.03 (s, 2H), 5.18 (s, 2H),
6.49 (d, 1H, J¼4.5 Hz), 6.83 (br s, 1H), 6.92 (m, 2H),
7.05 (br s, 1H), 7.32 (m, 15H), 7.47 (m, 2H), 7.56 (m,
1H), 7.80 (m, 4H), 7.94 (m, 2H), 10.88 (s, 1H). 13C NMR
(75 MHz, CD3OD): d 23.8, 24.8, 25.4, 28.7, 28.8, 28.9,
34.5, 35.1, 37.5, 38.0, 38.6, 38.7, 44.1, 45.5, 46.5, 47.4,
49.9, 65.4, 66.3, 69.2, 100.7, 105.3, 108.5, 117.4, 122.0,
122.9, 124.5, 127.4, 127.7, 127.9, 128.0, 128.5, 128.6,
129.8, 132.0, 132.6, 134.6, 137.2, 137.4, 137.9, 138.1,
147.8, 153.0, 155.3, 156.3, 170.2, 170.9, 171.3, 171.9.
FABHRMS calcd for C58H70N9O13S (M+H)+ 1132.4814,
found 1132.4833.
4.1.10. NPTX-8 (2). To a stirred solution of polyamine 17
(40 mg, 39.0 mmol) in DMF (1 mL) were added 2-mercap-
toethanol (10 mg, 128 mmol) and DBU (20 mg, 131 mmol).
After being stirred for 0.5 h at room temperature, the reac-
tion mixture was directly purified on silica gel column (2–
12% EtOH/CH2Cl2, then, 7.5% MeOH/CHCl3 containing
2.5% i-PrNH2) and then, further purification by preparative
TLC (7.5% MeOH/CHCl3 containing 2.5% i-PrNH2) gave
the secondary amine as a white powder.

K.-i. Nihei et al. / Tetrahedron 62 (2006) 8335–8350
8343
4.1.12. NPTX-1 (3). To a stirred solution of polyamine 18
(40 mg, 35.3 mmol) in DMF (1 mL) were added 2-mercap-
toethanol (9 mg, 115 mmol) and DBU (18 mg, 118 mmol).
After being stirred for 0.5 h at room temperature, the reac-
tion mixture was directly purified on silica gel column (2–
12% EtOH/CH2Cl2, then, 7.5% MeOH/CHCl3 containing
2.5% i-PrNH2) and then, further purification by preparative
TLC (7.5% MeOH/CHCl3 containing 2.5% i-PrNH2) gave
the secondary amine as a white powder.
TEA (156 mg, 1.54 mmol) slowly. After being stirred for
1 h at room temperature, the reaction mixture was diluted
with EtOAc (200 mL) and washed with saturated NH4Cl
solution (3ꢁ50 mL) and brine (3ꢁ50 mL). Aqueous layers
were extracted with EtOAc (3ꢁ20 mL) and combined
organic layers were dried over Na2SO4. Filtration and con-
centration followed by chromatography on silica gel (30–
40% EtOAc/hexane) gave the title compound (530 mg,
88%) as a colorless oil. IR (film) 1702, 1540, 1442, 1364,
1259, 1166, 854 cmꢂ1 1H NMR (300 MHz, CDCl3):
.
A solution of the secondary amine in AcOH (1 mL) was hy-
drogenated over 20% Pd(OH)2 on carbon (20 mg) for 2 h.
The mixture was filtrated through Celite and the filtrate
was evaporated to dryness. The resultant residue was dis-
solved in water and was purified by preparative RP-HPLC
with a linear gradient from 5% H2O/MeCN containing
0.1% TFA to 50% H2O/MeCN containing 0.1% TFA in
20 min. NPTX-1 (3) was eluted at 11.31 min and was
obtained as a colorless TFA salt (20 mg, 61%, two steps).
[a]2D2 +1.05 (c 1.14, H2O). IR (Nujol) 3513, 2924,
d 1.71 (quin, 2H, J¼5.7 Hz), 3.15 (q, 2H, J¼5.7 Hz), 3.28
(q, 2H, J¼5.7 Hz), 5.06 (br s, 1H), 5.08 (s, 2H), 5.86 (br s,
1H), 7.33 (m, 5H), 7.71 (m, 2H), 7.83 (m, 1H), 8.10 (m,
1H). 13C NMR (75 MHz, CDCl3): d 30.2, 37.6, 40.7, 66.8,
125.3, 128.0, 128.1, 128.5, 130.8, 132.7, 133.5, 133.8,
136.4, 148.0, 156.8. HRMS calcd for C11H15N2O2
(M+ꢂNs) 207.1133, found 207.1118.
4.1.15. 7-Benzyloxycarbonylamino-4-(2-nitrobenzene-
sulfonyl)-4-azaheptan-1-yl bromide (21). To a solution of
the above sulfonamide (420 mg, 1.07 mmol) and 1,3-dibro-
mopropane (648 mg, 3.21 mmol) in DMF (5 mL) was added
Cs2CO3 (525 mg, 1.61 mmol). After being stirred for 0.5 h
at 50 ꢀC, the reaction mixture was diluted with EtOAc
(100 mL) and washed with H2O (3ꢁ20 mL) and brine
(3ꢁ20 mL). Aqueous layers were extracted with EtOAc
(3ꢁ20 mL) and combined organic layers were dried over
Na2SO4. Filtration and concentration followed by chromato-
graphy on silica gel (20–50% EtOAc/hexane) gave the title
compound 21 (501 mg, 91%) as a colorless oil. IR (film)
1
1670 cmꢂ1. H NMR (300 MHz, D2O): d 0.90 (quin, 2H,
J¼7.2 Hz), 1.16 (m, 4H), 1.61 (m, 4H), 1.93 (quin, 2H,
J¼7.5 Hz), 2.46 (t, 2H, J¼6.6 Hz), 2.54 (dd, 1H, J¼6.0,
16.5 Hz), 2.60 (dd, 1H, J¼6.0, 16.5 Hz), 2.97 (m, 12H),
3.11 (t, 2H, J¼6.6 Hz), 3.68 (d, 1H, J¼15.9 Hz), 3.76 (d,
1H, J¼15.9 Hz), 4.46 (t, 1H, J¼6.0 Hz), 6.41 (t, 1H,
J¼4.2 Hz), 6.93 (d, 2H, J¼4.2 Hz), 7.06 (s, 1H). 13C
NMR (75 MHz, D2O): d 22.5, 22.6, 22.9, 23.6, 27.5, 27.6,
30.7, 33.8, 35.8, 36.4, 39.0, 39.1, 43.4, 44.4, 46.6, 46.9,
50.7, 103.5, 104.4, 106.6, 116.1, 122.9, 124.1, 138.6,
149.6, 171.4, 172.1, 174.5, 175.9. FABHRMS calcd for
C29H49N8O5 (M+H)+ 589.3810, found 589.3849. ESI-MS/
MS: Supplementary data 4.
1
1718, 1543, 1455, 1372, 1247, 1161, 852 cmꢂ1. H NMR
(300 MHz, CDCl3): d 1.79 (quin, 2H, J¼6.6 Hz), 2.08
(quin, 2H, J¼7.2 Hz), 3.23 (m, 2H), 3.38 (m, 6H), 5.09 (s,
2H), 5.14 (br s, 1H), 7.35 (m, 5H), 7.65 (m, 3H), 7.99 (m,
1H). 13C NMR (75 MHz, CDCl3): d 28.2, 29.8, 31.2, 37.7,
45.4, 46.1, 66.6, 124.3, 128.0, 128.1, 128.5, 130.9, 131.8,
132.8, 133.8, 136.5, 148.0, 156.4. HRMS calcd for
C14H20N2O2Br (M+ꢂNs) 327.0708, found 327.0710.
4.1.13. N-[Na-(tert-Butoxycarbonyl)-L-asparaginyl]-
N0-(2-nitrobenzenesulfonyl)-1,4-diaminobutane (19). To
a stirred solution of N-(2-nitrobenzenesulfonyl)-1,4-diami-
nobutane (500 mg, 1.83 mmol) in DMF (5 mL) was added
Boc-L-Asn-ONp (647 mg, 1.83 mmol) at 0 ꢀC. After being
stirred for 2 h at room temperature, the reaction mixture
was diluted with EtOAc (200 mL) and washed with satu-
rated aqueous NaHCO3 solution (5ꢁ50 mL) and brine
(3ꢁ50 mL). Aqueous layers were extracted with EtOAc
(3ꢁ40 mL) and combined organic layers were dried over
Na2SO4. Filtration and concentration followed by chromato-
graphy on silica gel (1–12% MeOH/CH2Cl2) gave the title
compound 19 (845 mg, 95%) as a white powder. [a]D22
ꢂ2.13 (c 2.44, DMF). IR (Nujol) 1681, 1659, 1543, 1461,
4.1.16. N-(Na-tert-Butoxycarbonyl-L-asparaginyl)-12-
benzyloxycarbonylamino-5,9-[di-(2-nitrobenzenesul-
fonyl)]-5,9-diaza-1-aminoundecane (22). To a solution of
sulfonamide 19 (244 mg, 500 mmol) and bromide 21
(387 mg, 752 mmol) in DMF (3 mL) were added Cs2CO3
(489 mg, 1.50 mmol) and TBAI (19.0 mg, 51.4 mmol). After
being stirred for 1 h at 70 ꢀC, the reaction mixture was
diluted with EtOAc (200 mL) and washed with H2O
(3ꢁ40 mL) and brine (3ꢁ40 mL). Aqueous layers were
extracted with EtOAc (3ꢁ40 mL) and combined organic
layers were dried over Na2SO4. Filtration and concentration
followed by chromatography on silica gel (2–4% MeOH/
CH2Cl2) gave the title compound 22 (433 mg, 94% from
19) as a colorless solid. [a]2D4 ꢂ2.43 (c 1.48, MeOH). IR
1
1161 cmꢂ1. H NMR (300 MHz, DMSO-d6): d 1.35 (m,
13H), 2.34 (m, 2H), 2.86 (m, 2H), 2.96 (quin, 2H,
J¼6.0 Hz), 4.13 (m, 1H), 6.79 (d, 1H, J¼8.1 Hz), 6.86 (br
s, 1H), 7.24 (br s, 1H), 7.69 (br s, 1H), 7.83 (m, 2H), 7.92
(m, 2H), 8.04 (m, 1H). 13C NMR (75 MHz, CD3OD):
d 26.3, 26.6, 28.4, 37.7, 38.2, 42.6, 51.7, 78.4, 124.6,
129.6, 132.8, 133.0, 134.2, 148.0, 155.3, 171.5, 175.8.
HRMS calcd for C14H20N5O6S (M+ꢂBoc) 386.1134, found
386.1113.
(Nujol) 1712, 1670, 1544, 1459, 1372, 1161, 852 cmꢂ1
.
1H NMR (300 MHz, CDCl3): d 1.44 (s, 9H), 1.53 (m, 4H),
1.78 (m, 4H), 2.51 (dd, 1H, J¼6.0, 15.6 Hz), 2.88 (dd, 1H,
J¼4.8, 15.6 Hz), 3.25 (m, 12H), 4.41 (m, 1H), 5.08 (s,
2H), 5.50 (br s, 1H), 5.68 (br s, 1H), 6.08 (br s, 2H), 6.85
(br s, 1H), 7.35 (m, 5H), 7.61 (m, 2H), 7.68 (m, 4H), 7.96
(m, 2H). 13C NMR (75 MHz, CDCl3): d 25.1, 26.7, 27.6,
28.3, 28.4, 38.0, 38.4, 45.1, 45.4, 45.5, 47.6, 51.2, 66.6,
80.3, 124.2, 124.3, 128.1, 128.5, 130.6, 131.8, 131.9,
132.7, 132.9, 133.6, 133.7, 136.6, 148.0, 155.7, 156.6,
4.1.14. N-(2-Nitrobenzenesulfonyl)-N0-(benzyloxycar-
bonyl)-1,3-diaminopropane. To a cold (0 ꢀC) and stirred
suspension of N-(2-nitrobenzenesulfonyl)-1,3-diaminopro-
pane22 (20) (400 mg, 1.54 mmol) in CH2Cl2 (10 mL) were
added CbzCl (30% in toluene, 876 mg, 1.54 mmol) and

8344
K.-i. Nihei et al. / Tetrahedron 62 (2006) 8335–8350
171.4, 173.4. FABHRMS calcd for C39H52N8O14S2Na
(M+Na)+ 943.2942, found 943.2951.
1.87 (quin, 2H, J¼8.1 Hz), 1.93 (quin, 2H, J¼6.6 Hz),
2.56 (dd, 1H, J¼8.1, 15.3 Hz), 2.64 (dd, 1H, J¼7.2,
15.3 Hz), 2.78 (m, 4H), 2.98 (m, 8H), 3.69 (s, 2H), 4.47
(dd, 1H, J¼7.2, 8.1 Hz), 7.06 (t, 1H, J¼8.1 Hz), 7.16 (t,
1H, J¼8.1 Hz), 7.22 (s, 1H), 7.41 (d, 1H, J¼8.1 Hz), 7.48
(d, 1H, J¼8.1 Hz). 13C NMR (75 MHz, D2O): d 22.6,
22.7, 23.9, 25.5, 32.4, 36.2, 36.7, 38.6, 44.1, 44.7, 45.5,
47.2, 51.3, 107.8, 112.2, 118.6, 119.8, 122.4, 125.2, 126.8,
136.5, 172.8, 174.6, 175.4. FABHRMS calcd for
C24H39N7O3 (M+H)+ 474.3193, found 474.3171. ESI-MS/
MS: Supplementary data 5.
4.1.17. N-[Na-(Indoleacetyl)-L-asparaginyl]-12-benzyl-
oxycarbonylamino-5,9-[di-(2-nitrobenzenesulfonyl)]-
5,9-diaza-1-aminoundecane (23). To a cold (0 ꢀC) and
stirred suspension of polyamine 22 (156 mg, 170 mmol) in
CHCl3 (1 mL) was added TFA (1 mL) slowly. After being
stirred for 1 h at room temperature, the reaction mixture
was concentrated in vacuo to give the crude amine TFA
salt as a colorless solid.
To a stirred solution of obtained amine TFA salt in DMF
(1 mL) were added TEA (18 mg, 178 mmol) and indoleace-
tic acid succinimide ester45 (70 mg, 257 mmol) slowly. After
being stirred for 2 h at room temperature, the reaction mix-
ture was diluted with EtOAc (100 mL) and washed with sat-
urated NaHCO3 solution (3ꢁ20 mL) and brine (3ꢁ20 mL).
Aqueous layers were extracted with EtOAc (3ꢁ20 mL) and
combined organic layers were dried over Na2SO4. Filtration
and concentration followed by chromatography on silica gel
(2–8% EtOH/CH2Cl2) gave the title compound 23 (139 mg,
84%, two steps) as a white powder. [a]2D2 +5.06 (c 1.76, ace-
tone). IR (Nujol) 1666, 1542, 1461, 1376, 1256, 1160,
4.1.19. N-(2-Nitrobenzenesulfonyl)-N0-(benzyloxycar-
bonyl)-1,4-diaminobutane. To a cold (0 ꢀC) and stirred sus-
pension of N-(2-nitrobenzenesulfonyl)-1,4-diaminobutane22
(12) (400 mg, 1.46 mmol) in CH2Cl2 (10 mL) were added
CbzCl (30% in toluene, 830 mg, 1.46 mmol) and TEA
(148 mg, 1.46 mmol) slowly. After being stirred for 1 h at
room temperature, the reaction mixture was diluted with
EtOAc (200 mL) and washed with saturated NH4Cl solution
(3ꢁ50 mL) and brine (3ꢁ50 mL). Aqueous layers were ex-
tracted with EtOAc (3ꢁ20 mL) and combined organic layers
were dried over Na2SO4. Filtration and concentration fol-
lowed by chromatography on silica gel (30–50% EtOAc/
hexane) gave the title compound (564 mg, 95%) as a color-
less oil. IR (film) 1703, 1540, 1442, 1364, 1252, 1166,
852 cmꢂ1 1H NMR (300 MHz, CD3COCD3): d 1.21
.
(quin, 2H, J¼6.3 Hz), 1.39 (quin, 2H, J¼7.2 Hz), 1.75
(quin, 2H, J¼6.9 Hz), 1.78 (quin, 2H, J¼6.6 Hz), 2.52 (dd,
1H, J¼6.0, 15.6 Hz), 2.72 (dd, 1H, J¼5.1, 15.6 Hz), 3.02
(m, 2H), 3.13 (m, 2H), 3.20 (m, 2H), 3.23 (m, 2H), 3.30
(m, 2H), 3.37 (m, 2H), 3.69 (s, 2H), 4.65 (m, 1H), 5.06 (s,
2H), 6.31 (br s, 1H), 6.43 (br s, 1H), 6.94 (br s, 1H), 7.01
(t, 1H, J¼6.9 Hz), 7.09 (t, 1H, J¼6.9 Hz), 7.33 (m, 7H),
7.53 (br s, 1H), 7.59 (d, 1H, J¼6.9 Hz), 7.83 (m, 6H), 8.00
(m, 3H), 10.13 (br s, 1H). 13C NMR (75 MHz, CD3COCD3):
d 25.1, 26.6, 27.2, 33.3, 36.7, 38.1, 38.2, 38.4, 44.8, 45.3,
45.7, 47.4, 50.2, 65.9, 109.2, 111.7, 118.8, 119.2, 121.8,
124.1, 124.3, 124.4, 124.5, 127.8, 128.0, 128.1, 128.6,
130.4, 130.5, 132.4, 132.5, 132.9, 133.1, 134.3, 134.5,
137.0, 137.8, 148.4, 156.7, 171.9, 172.3, 173.8. FABHRMS
calcd for C44H51N9O13S2Na (M+Na)+ 1000.2946, found
1000.2936.
1
854 cmꢂ1. H NMR (300 MHz, CDCl3): d 1.55 (m, 4H),
3.10 (m, 2H), 3.16 (m, 2H), 4.89 (br s, 1H), 5.08 (s, 2H),
5.46 (br s, 1H), 7.35 (m, 5H), 7.72 (m, 2H), 7.84 (m, 1H),
8.12 (m, 1H). 13C NMR (75 MHz, CDCl3): d 26.8, 26.9,
40.3, 43.3, 66.6, 125.3, 128.0, 128.5, 131.0, 132.8, 133.6,
136.5, 148.0, 156.5. HRMS calcd for C12H17N2O2
(M+ꢂNs) 221.1290, found 221.1299.
4.1.20. Methyl 7-benzyloxycarbonylamino-3-(2-nitro-
benzenesulfonyl)-3-azaheptanoate. To a solution of the
above sulfonamide (514 mg, 1.26 mmol) and methyl bromo-
acetate (289 mg, 1.89 mmol) in DMF (4 mL) was added
Cs2CO3 (616 mg, 1.89 mmol). After being stirred for 0.5 h
at 50 ꢀC, the reaction mixture was diluted with EtOAc
(150 mL) and washed with H2O (3ꢁ30 mL) and brine
(3ꢁ30 mL). Aqueous layers were extracted with EtOAc
(3ꢁ30 mL) and combined organic layers were dried over
Na2SO4. Filtration and concentration followed by chromato-
graphy on silica gel (30–50% EtOAc/hexane) gave the ester
(520 mg, 86%) as a colorless oil. IR (film) 1752, 1714, 1546,
4.1.18. NPTX-473 (4). To a stirred solution of polyamine 23
(40 mg, 40.9 mmol) in DMF (1 mL) were added 2-mercap-
toethanol (20 mg, 256 mmol) and DBU (40 mg, 263 mmol).
After being stirred for 0.5 h at room temperature, the reac-
tion mixture was directly purified on silica gel column (2–
12% EtOH/CH2Cl2, then, 7.5% MeOH/CHCl3 containing
2.5% i-PrNH2) and then, further purification by preparative
TLC (7.5% MeOH/CHCl3 containing 2.5% i-PrNH2) gave
the secondary amine as a white powder.
1439, 1372, 1254, 1218, 1164, 853 cmꢂ1 1H NMR
.
(300 MHz, CDCl3): d 1.54 (m, 4H), 3.17 (m, 2H), 3.41 (t,
2H, J¼7.2 Hz), 3.65 (s, 3H), 4.16 (s, 2H), 5.00 (br s, 1H),
5.08 (s, 2H), 7.35 (m, 5H), 7.60 (m, 1H), 7.68 (m, 2H),
8.06 (m, 1H). 13C NMR (75 MHz, CDCl3): d 24.8, 26.8,
40.3, 47.6, 48.1, 52.3, 66.6, 124.1, 128.0, 128.1, 128.5,
130.8, 131.7, 133.2, 133.6, 136.6, 147.9, 156.5, 162.8.
HRMS calcd for C13H18N3O6S (M+ꢂCbz) 344.0917, found
344.0918.
A solution of the secondary amine in AcOH (1 mL) was hy-
drogenated over 20% Pd(OH)2 on carbon (20 mg) for 2 h.
The mixture was filtrated through Celite and the filtrate
was evaporated to dryness. The resultant residue was dis-
solved in water and was purified by preparative RP-HPLC
with a linear gradient from 5% H2O/MeCN containing
0.1% TFA to 50% H2O/MeCN containing 0.1% TFA in
20 min. NPTX-473 (4) was eluted at 12.02 min and was ob-
tained as a colorless TFA salt (20 mg, 60%, two steps). [a]D22
ꢂ3.81 (c 0.42, H2O). IR (Nujol) 3300, 2924, 1670,
4.1.21. Methyl 7-benzyloxycarbonylamino-3-(2-nitro-
benzenesulfonyl)-3-azaheptanoic acid (24). To a cold
(0 ꢀC) and stirred solution of the above ester (500 mg,
1.04 mmol) in MeOH (5 mL) was added 3.0 M aqueous
NaOH solution (1.04 mL, 3.12 mmol) slowly. After being
stirred for 2 h at room temperature, the reaction mixture
was adjusted to pH 2.0 by concentrated HCl. The resultant
1
1549 cmꢂ1. H NMR (300 MHz, D2O): d 1.32 (m, 4H),

K.-i. Nihei et al. / Tetrahedron 62 (2006) 8335–8350
8345
mixture was diluted with EtOAc (100 mL) and washed with
H2O (3ꢁ20 mL) and brine (3ꢁ20 mL). Aqueous layers were
extracted with EtOAc (3ꢁ20 mL) and combined organic
layers were dried over Na2SO4. Filtration and concentration
followed by chromatography on silica gel (2–40% MeOH/
CH2Cl2) gave the title compound 24 (414 mg, 86%) as a col-
orless oil. IR (film) 1729, 1714, 1548, 1455, 1371, 1259,
(2–10% EtOH/CH2Cl2) gave the title compound 26
(113 mg, 73%, four steps) as a white powder. [a]2D2 +5.59
(c 2.22, DMF). IR (Nujol) 1660, 1538, 1456, 1376, 1257,
1
1158, 852 cmꢂ1. H NMR (300 MHz, DMSO-d6): d 1.14
(m, 2H), 1.28 (m, 6H), 1.45 (m, 2H), 2.37 (dd, 1H, J¼7.8,
15.3 Hz), 2.48 (dd, 1H, J¼6.0, 15.3 Hz), 2.94 (m, 6H),
3.27 (m, 2H), 3.54 (s, 2H), 3.93 (s, 2H), 4.50 (dd, 1H,
J¼6.0, 7.8 Hz), 4.98 (s, 2H), 6.84 (br s, 1H), 6.94 (t, 1H,
J¼6.9 Hz), 7.05 (t, 1H, J¼6.9 Hz), 7.18 (br s, 1H), 7.21 (t,
1H, J¼5.4 Hz), 7.33 (m, 7H), 7.51 (d, 1H, J¼8.1 Hz), 7.59
(t, 1H, J¼5.4 Hz), 7.80 (m, 2H), 7.91 (m, 2H), 8.10 (m,
2H), 10.84 (s, 1H). 13C NMR (75 MHz, DMSO-d6):
d 23.7, 24.7, 26.7, 28.8, 32.7, 37.5, 38.7, 48.2, 48.7, 50.1,
65.4, 108.9, 111.5, 118.5, 118.9, 121.2, 124.0, 124.3,
127.4, 127.9, 128.0, 128.6, 130.3, 132.3, 132.4, 134.5,
136.3, 137.5, 147.8, 156.3, 167.1, 170.8, 170.9, 171.7.
FABHRMS calcd for C39H48N8O10SNa (M+Na)+
843.3112, found 843.3115.
1
1162, 853 cmꢂ1. H NMR (300 MHz, CDCl3): d 1.34 (m,
2H), 1.41 (m, 2H), 3.02 (m, 2H), 3.31 (m, 2H), 4.04 (s,
2H), 5.02 (s, 2H), 5.07 (br s, 1H), 7.29 (m, 5H), 7.54 (m,
3H), 8.00 (m, 1H). 13C NMR (75 MHz, CDCl3): d 24.5,
26.6, 40.3, 48.1, 49.0, 66.6, 124.1, 127.9, 128.0, 128.4,
130.7, 132.0, 133.0, 133.5, 136.5, 147.7, 156.6, 174.4.
HRMS calcd for C14H19N2O4 (M+ꢂNs) 279.1345, found
279.1347.
4.1.22. N-[Na-(Indoleacetyl)-L-asparaginyl]-N0-[7-benzyl-
oxycarbonylamino-3-(2-nitrobenzenesulfonyl)-3-aza-
heptanoyl]-1,5-diaminopentane (26). To a cold (0 ꢀC) and
stirred suspension of carboxylic acid 24 (186 mg, 400 mmol)
and HOSu (92 mg, 799 mmol) in CH2Cl2 (5 mL) was added
DCC (124 mg, 601 mmol) slowly. After being stirred for 4 h
at 0 ꢀC, the reaction mixture was filtrated through a pad
of silica gel with EtOAc. The filtrate was concentrated
in vacuo to give the crude succinimidyl ester as a colorless
oil.
4.1.23. NPTX-501 (5). To a stirred solution of polyamine 26
(40 mg, 48.7 mmol) in DMF (1 mL) were added 2-mercap-
toethanol (12 mg, 154 mmol) and DBU (24 mg, 158 mmol).
After being stirred for 0.5 h at room temperature, the reac-
tion mixture was directly purified on silica gel column (2–
12% EtOH/CH2Cl2, then, 7.5% MeOH/CHCl3 containing
2.5% i-PrNH2) and then, further purification by preparative
TLC (7.5% MeOH/CHCl3 containing 2.5% i-PrNH2) gave
the secondary amine as a white powder.
To a stirred solution of obtained ester in DMF (3 mL) was
added amine 11 (127 mg, 401 mmol) slowly. After being
stirred for 0.5 h at room temperature, the reaction mixture
was diluted with EtOAc (100 mL) and washed with satu-
rated aqueous NaHCO3 solution (3ꢁ20 mL) and brine
(3ꢁ20 mL). Aqueous layers were extracted with EtOAc
(3ꢁ20 mL) and combined organic layers were dried over
Na2SO4. Filtration and concentration followed by chromato-
graphy on silica gel (2–8% MeOH/CH2Cl2) gave the poly-
amine 25 (252 mg), which was used in the next step
without further purification. 1H NMR (300 MHz, CD3OD):
d 1.29 (m, 2H), 1.43 (s, 9H), 1.45 (m, 8H), 2.57 (dd, 1H,
J¼6.3, 13.2 Hz), 2.66 (dd, 1H, J¼6.3, 13.2 Hz), 3.06 (t,
2H, J¼6.6 Hz), 3.12 (t, 2H, J¼6.0 Hz), 3.15 (t, 2H,
J¼6.6 Hz), 3.39 (t, 2H, J¼6.6 Hz), 4.02 (s, 2H), 4.36 (t,
1H, J¼6.3 Hz), 5.04 (s, 2H), 7.32 (m, 5H), 7.75 (m, 3H),
8.10 (m, 1H). 13C NMR (75 MHz, CD3OD): d 25.0, 26.0,
27.9, 28.7, 29.9, 38.4, 40.3, 41.2, 50.4, 53.1, 67.3, 80.9,
125.4, 128.8, 129.0, 129.5, 131.9, 133.1, 133.9, 135.3,
138.5, 149.5, 157.5, 158.9, 170.2, 173.8, 175.1.
A solution of the secondary amine in AcOH (1 mL) was hy-
drogenated over 20% Pd(OH)2 on carbon (20 mg) for 2 h.
The mixture was filtrated through Celite and the filtrate
was evaporated to dryness. The resultant residue was dis-
solved in water and was purified by preparative RP-HPLC
with a linear gradient from 5% H2O/MeCN containing
0.1% TFA to 50% H2O/MeCN containing 0.1% TFA in
20 min. NPTX-501 (5) was eluted at 13.21 min and was ob-
tained as a colorless TFA salt (23 mg, 65%, two steps). [a]D22
ꢂ0.41 (c 1.22, H2O). IR (Nujol) 3554, 2924, 1698,
1
1541 cmꢂ1. H NMR (300 MHz, D2O): d 0.99 (quin, 2H,
J¼7.2 Hz), 1.23 (m, 4H), 1.55 (m, 4H), 2.51 (dd, 1H,
J¼7.8, 15.3 Hz), 2.60 (dd, 1H, J¼6.0, 15.3 Hz), 2.84 (m,
4H), 2.97 (m, 4H), 3.56 (s, 2H), 3.66 (s, 2H), 4.48 (dd, 1H,
J¼6.0, 7.8 Hz), 7.03 (t, 1H, J¼8.1 Hz), 7.13 (t, 1H,
J¼8.1 Hz), 7.19 (s, 1H), 7.38 (d, 1H, J¼8.1 Hz), 7.44 (d,
1H, J¼8.1 Hz). 13C NMR (75 MHz, D2O): d 22.4, 22.9,
23.7, 27.6, 32.1, 36.1, 38.6, 39.0, 39.1, 46.8, 47.7, 50.8,
107.4, 111.9, 118.2, 119.5, 122.1, 124.8, 126.5, 136.2,
165.4, 172.1, 174.4, 174.9. FABHRMS calcd for
C25H40N7O4 (M+H)+ 502.3142, found 502.3108. ESI-MS/
MS: Supplementary data 6.
To a cold (0 ꢀC) and stirred suspension of polyamine 25
(120 mg, 157 mmol) in CHCl3 (1 mL) was added TFA
(1 mL) slowly. After being stirred for 1 h at room tempera-
ture, the reaction mixture was concentrated in vacuo to
give the crude amine TFA salt as a colorless solid.
4.1.24. N-[Na-(tert-Butoxycarbonyl)-L-asparaginyl]-N0-
[8-(Na,Nd,Nu-tribenzyloxycarbonylarginyl)-amino-4-
benzyloxycarbonyl-4-azaoctanoyl]-1,5-diaminopentane
(27). To a stirred solution of polyamine 14 (300 mg,
386 mmol) in DMF (1 mL) were added 2-mercaptoethanol
(91 mg, 1.16 mmol) and DBU (176 mg, 1.16 mmol). After
being stirred for 0.5 h at room temperature, the reaction mix-
ture was directly subjected to silica gel chromatography (2–
12% EtOH/CH2Cl2, then, 7.5% MeOH/CHCl3 containing
2.5% i-PrNH2) to afford the secondary amine as a white
powder.
To a stirred solution of obtained amine TFA salt in DMF
(1 mL) were added TEA (16 mg, 158 mmol) and indoleace-
tic acid succinimide ester54 (65 mg, 239 mmol) slowly. After
being stirred for 2 h at room temperature, the reaction mix-
ture was diluted with EtOAc (100 mL) and washed with sat-
urated NaHCO3 solution (3ꢁ20 mL) and brine (3ꢁ20 mL).
Aqueous layers were extracted with EtOAc (3ꢁ20 mL) and
combined organic layers were dried over Na2SO4. Filtration
and concentration followed by chromatography on silica gel

8346
K.-i. Nihei et al. / Tetrahedron 62 (2006) 8335–8350
To a stirred solution of obtained amine in DMF (1 mL) was
added tri-Cbz-arginine succinimide ester48 (389 mg,
577 mmol) slowly. After being stirred for 1 h at room temper-
ature, the reaction mixture was directly subjected to silica
gel chromatography (2–8% EtOH/CH2Cl2) to afford the title
compound 27 (337 mg, 76%) as a white powder. [a]2D2 +6.36
(c 1.32, DMSO). IR (Nujol) 1725, 1688, 1652, 1259,
MeCN containing 0.1% TFA in 20 min. NSTX-3 (6) was
eluted at 9.66 min and was obtained as a colorless TFA
salt (14 mg, 74%). [a]D22 +2.95 (c 1.73, H2O). IR (Nujol)
1
3444, 2925, 1696, 1523 cmꢂ1. H NMR (300 MHz, D2O):
d 1.03 (quin, 2H, J¼7.5 Hz), 1.29 (m, 4H), 1.52 (m, 6H),
1.77 (dt, 2H, J¼9.6, 6.9 Hz), 2.50 (t, 2H, J¼6.9 Hz), 2.52
(dd, 1H, J¼7.8, 15.0 Hz), 2.63 (dd, 1H, J¼5.7, 15.0 Hz),
2.93 (t, 4H, J¼8.1 Hz), 3.08 (t, 4H, J¼6.9 Hz), 3.13 (t,
4H, J¼6.9 Hz), 3.34 (d, 1H, J¼15.3 Hz), 3.43 (d, 1H,
J¼15.3 Hz), 3.81 (t, 1H, J¼6.9 Hz), 4.46 (dd, 1H, J¼5.7,
7.8 Hz), 6.31 (m, 2H), 6.94 (d, 1H, J¼9.0 Hz). 13C NMR
(75 MHz, D2O): d 22.8, 23.0, 23.5, 25.3, 27.6, 27.7, 27.9,
30.8, 35.9, 36.8, 38.7, 39.1, 39.2, 40.2, 43.3, 46.9, 50.8,
52.9, 102.7, 107.4, 113.6, 132.3, 155.1, 156.1, 156.7,
169.2, 171.5, 172.2, 174.6. FABHRMS calcd for
C30H53N10O7 (M+H)+ 665.4107, found 665.4083. ESI-
MS/MS: Supplementary data 7.
1
1103 cmꢂ1. H NMR (300 MHz, DMSO-d6): d 1.25 (m,
2H), 1.35 (m, 19H), 1.55 (m, 4H), 2.29 (t, 2H, J¼6.9 Hz),
2.35 (m, 2H), 2.98 (m, 6H), 3.15 (m, 2H), 3.42 (m, 2H),
3.84 (m, 3H), 4.16 (dd, 1H, J¼5.2, 6.6 Hz), 4.97 (s, 2H),
5.03 (s, 4H), 5.20 (s, 2H), 6.81 (d, 1H, J¼8.4 Hz), 6.87 (s,
1H), 7.31 (m, 20H), 7.67 (br s, 1H), 7.85 (m, 2H), 9.14 (br
s, 2H). 13C NMR (75 MHz, DMSO-d6): d 23.8, 25.3, 26.5,
28.4, 28.9, 29.5, 34.4, 35.2, 37.6, 38.5, 38.7, 43.3, 44.0,
44.6, 46.6, 46.7, 51.7, 54.8, 65.6, 66.2, 66.3, 68.4, 78.4,
127.5, 127.8, 127.9, 128.1, 128.4, 128.5, 128.6, 128.7,
135.5, 137.2, 137.3, 155.2, 155.3, 155.4, 156.1, 159.9,
163.1, 170.2, 171.4, 171.7, 171.8. FABHRMS calcd for
C59H79N10O14 (M+H)+ 1151.5778, found 1151.5778.
4.1.27. Methyl 7-(2-nitrobenzenesulfonyl)-amino-4-ben-
zyloxycarbonyl-4-azahenpanoate. A solution of methyl
acrylate (166 mg, 1.93 mmol) in EtOH (10 mL) was added
at room temperature over 5 h to a stirring solution of N-(2-
nitrobenzenesulfonyl)-1,3-diaminopropane18 (20) (500 mg,
1.93 mmol) in EtOH (20 mL). The solvent was evaporated
in vacuo, and the residue was subjected to silica gel chroma-
tography (5–20% MeOH/CH2Cl2) to give the crude second-
ary amine as a colorless oil.
4.1.25. N-[Na-(tert-Butoxycarbonyl)-L-asparaginyl]-N0-
[8-(Na,Nd,Nu-tribenzyloxycarbonylarginyl)-amino-4-
benzyloxycarbonyl-4-azaoctanoyl]-1,5-diaminopentane
(28). To a cold (0 ꢀC) and stirred suspension of polyamine 27
(100 mg, 86.9 mmol) in CHCl3 (1 mL) was added TFA
(1 mL) slowly. After being stirred for 1 h at room tempera-
ture, the reaction mixture was concentrated in vacuo to
give the crude amine TFA salt as a colorless solid.
To a cold (0 ꢀC) and stirred solution of obtained secondary
amine in CH2Cl2 (20 mL) were added CbzCl (30% in tolu-
ene, 1.10 g, 1.93 mmol) and Et3N (195 mg, 1.93 mmol)
slowly. After being stirred for 2 h at room temperature, the
reaction mixture was diluted with EtOAc (200 mL) and
washed with saturated NH4Cl solution (3ꢁ50 mL) and brine
(3ꢁ50 mL). Aqueous layers were extracted with EtOAc
(3ꢁ20 mL) and combined organic layers were dried over
Na2SO4. Filtration and concentration followed by chromato-
graphy on silica gel (20–50% EtOAc/hexane) gave the
methyl ester (796 mg, 86%, two steps) as a colorless oil.
To a stirred solution of obtained amine TFA salt in DMF
(1 mL) were added TEA (9 mg, 89.0 mmol) and 2,4-di-
benzyloxyphenylacetic acid succinimide ester28 (58 mg,
130 mmol) slowly. After being stirred for 2 h at room temper-
ature, the reaction mixture was directly subjected to silica
gel chromatography (2–8% MeOH/CH2Cl2) to afford the ti-
tle compound 28 (104 mg, 87%) as a white powder. [a]D22
+2.57 (c 1.05, DMF). IR (Nujol) 1691, 1664, 1645, 1614,
1
1260 cmꢂ1. H NMR (300 MHz, DMSO-d6): d 1.17 (m,
2H), 1.31 (m, 8H), 1.50 (m, 4H), 2.29 (t, 2H, J¼6.6 Hz),
2.36 (dd, 1H, J¼6.3, 15.9 Hz), 2.43 (dd, 1H, J¼6.0,
15.9 Hz), 2.96 (m, 6H), 3.14 (m, 2H), 3.27 (m, 2H), 3.41
(s, 2H), 3.84 (m, 3H), 4.53 (m, 1H), 4.97 (s, 2H), 5.03 (s,
6H), 5.08 (s, 2H), 5.20 (s, 2H), 6.52 (d, 1H, J¼9.3 Hz),
6.68 (s, 1H), 6.85 (s, 1H), 7.07 (d, 1H, J¼8.4 Hz), 7.35
(m, 32H), 7.60 (t, 1H, J¼7.2 Hz), 7.83 (m, 2H), 7.96 (d,
1H, J¼8.1 Hz)), 9.14 (br s, 2H). 13C NMR (75 MHz,
DMSO-d6): d 22.8, 23.8, 25.3, 26.5, 28.8, 28.9, 29.5, 36.4,
37.5, 38.5, 38.6, 38.7, 44.6, 46.5, 49.4, 50.0, 54.7, 65.6,
66.2, 66.3, 68.3, 69.4, 69.5, 100.7, 105.9, 117.3, 127.3,
127.5, 127.9, 128.0, 128.6, 128.7, 131.1, 135.5, 137.2,
137.3, 137.4, 155.1, 155.3, 156.1, 157.1, 158.5, 159.9,
163.1, 170.1, 170.5, 170.8, 171.6, 171.7. FABHRMS calcd
for C76H88N10O15Na (M+Na)+ 1403.6328, found
1403.6323.
IR (film) 1732, 1695, 1539, 1480, 1368, 1125, 854 cmꢂ1
.
1H NMR (300 MHz, CDCl3): d 1.73 (quin, 2H, J¼6.6 Hz),
2.57 (m, 2H), 3.11 (m, 2H), 3.37 (t, 2H, J¼6.3 Hz), 3.49
(t, 2H, J¼7.2 Hz), 3.64 (s, 3H), 5.12 (s, 2H), 5.44 (br s,
1H), 7.33 (m, 5H), 7.72 (m, 2H), 7.83 (m, 1H), 8.09 (m,
1H). 13C NMR (75 MHz, CDCl3): d 28.5, 33.6, 40.6, 43.1,
44.7, 51.8, 67.4, 125.2, 127.9, 128.1, 128.5, 130.7, 132.6,
133.3, 134.2, 136.3, 148.0, 155.6, 171.9. HRMS calcd for
C20H22N3O7S (M+ꢂOMe) 448.1179, found 448.1183.
4.1.28. 7-(2-Nitrobenzenesulfonyl)-amino-4-benzyloxy-
carbonyl-4-azahenpanoic acid (29). To a cold (0 ꢀC) and
stirred solution of the above ester (600 mg, 1.25 mmol) in
MeOH (4 mL) was added 3.0 M aqueous NaOH solution
(1.25 mL, 3.75 mmol) slowly. After being stirred for 1 h at
room temperature, the reaction mixture was adjusted to pH
2.0 by concentrated HCl. The resultant mixture was diluted
with EtOAc (200 mL) and washed with H2O (3ꢁ50 mL) and
brine (3ꢁ50 mL). Aqueous layers were extracted with
EtOAc (3ꢁ40 mL) and combined organic layers were dried
over Na2SO4. Filtration and concentration followed by chro-
matography on silica gel (1–40% MeOH/CH2Cl2) gave
the title compound 29 (494 mg, 85%) as a colorless oil.
4.1.26. NSTX-3 (6). A solution of the polyamine 28 (26 mg,
18.8 mmol) in AcOH (1 mL) was hydrogenated over 20%
Pd(OH)2 on carbon (20 mg) for 4 h. The mixture was fil-
trated through Celite and the filtrate was evaporated to dry-
ness. The resultant residue was dissolved in water and was
purified by preparative RP-HPLC with a linear gradient
from 5% H2O/MeCN containing 0.1% TFA to 50% H2O/
IR (film) 1694, 1540, 1484, 1424, 1365, 1166, 854 cmꢂ1
.

K.-i. Nihei et al. / Tetrahedron 62 (2006) 8335–8350
8347
1H NMR (300 MHz, CDCl3): d 1.75 (quin, 2H, J¼6.6 Hz),
2.59 (m, 2H), 3.09 (m, 2H), 3.37 (m, 2H), 3.50 (t, 2H,
J¼6.6 Hz), 5.12 (s, 2H), 5.47 (br s, 1H), 7.33 (m, 5H),
7.70 (m, 2H), 7.83 (m, 1H), 8.08 (m, 1H). 13C NMR
(75 MHz, CDCl3): d 28.4, 33.4, 40.7, 42.9, 43.7, 67.4,
125.2, 127.8, 128.0, 128.5, 130.7, 132.6, 133.3, 136.2,
147.9, 156.5, 175.9. HRMS calcd for C12H16N3O6S
(M+ꢂNa) 330.0760, found 330.0757.
4.1.29. N-[Na-(tert-Butoxycarbonyl)-L-asparaginyl]-N0-
[7-(2-nitrobenzenesulfonylamino)-4-benzyloxycarbonyl-
4-azaheptanoyl]-1,5-diaminopentane (30). To a cold
(0 ꢀC) and stirred suspension of carboxylic acid 29
(377 mg, 810 mmol) and HOSu (186 mg, 1.62 mmol) in
CH2Cl2 (5 mL) was added DCC (252 mg, 1.22 mmol)
slowly. After being stirred for 5 h at 0 ꢀC, the reaction mix-
ture was filtrated through a pad of silica gel with EtOAc. The
filtrate was concentrated in vacuo to give the crude succini-
midyl ester as a colorless oil.
CH2Cl2) gave the title compound 31 (174 mg, 88%, two
steps) as a white powder. [a]2D2 ꢂ3.88 (c 2.55, DMF). IR
(Nujol) 1641, 1542, 1457, 1376, 1239, 1164, 853 cmꢂ1
.
1H NMR (300 MHz, DMSO-d6): d 1.14 (m, 2H), 1.32 (m,
4H), 1.62 (m, 2H), 2.27 (t, 2H, J¼7.2 Hz), 2.34 (dd, 1H,
J¼7.8, 15.6 Hz), 2.43 (dd, 1H, J¼6.3, 15.6 Hz), 2.87 (m,
2H), 2.96 (m, 4H), 3.18 (m, 2H), 3.36 (m, 4H), 4.47 (dd,
1H, J¼6.3, 7.8 Hz), 5.02 (s, 2H), 5.05 (s, 2H), 6.85 (br s,
1H), 6.90 (d, 2H, J¼8.4 Hz), 7.14 (d, 2H, J¼8.4 Hz), 7.32
(m, 10H), 7.41 (m, 1H), 7.67 (t, 1H, J¼5.7 Hz), 7.84 (m,
3H), 7.95 (m, 2H), 8.07 (br s, 1H), 8.15 (d, 1H, J¼8.4 Hz).
13C NMR (75 MHz, CDCl3): d 23.8, 28.0, 28.8, 28.9, 34.3,
35.0, 37.6, 38.6, 38.7, 40.7, 41.4, 43.5, 44.8, 50.1, 66.3,
69.4, 114.7, 124.6, 127.5, 127.8, 127.9, 128.0, 128.5,
128.6, 129.6, 130.3, 132.7, 132.8, 134.3, 137.2, 137.4,
148.0, 157.1, 170.1, 170.5, 170.9, 171.6. FABHRMS calcd
for C44H54N7O11S (M+H)+ 888.3602, found 888.3604.
4.1.31. Joramine (7). To a stirred solution of polyamine 31
(40 mg, 45.1 mmol) in DMF (1 mL) were added 2-mercap-
toethanol (11 mg, 141 mmol) and DBU (22 mg, 145 mmol).
After being stirred for 0.5 h at room temperature, the reac-
tion mixture was directly purified on silica gel column (2–
12% EtOH/CH2Cl2, then, 7.5% MeOH/CHCl3 containing
2.5% i-PrNH2) and then, further purification by preparative
TLC (7.5% MeOH/CHCl3 containing 2.5% i-PrNH2) gave
the primary amine as a white powder.
To a stirred solution of obtained ester in DMF (6 mL) was
added amine 11 (256 mg, 0.81 mmol) slowly. After being
stirred for 0.5 h at room temperature, the reaction mixture
was diluted with EtOAc (100 mL) and washed with satu-
rated aqueous NaHCO3 solution (3ꢁ20 mL) and brine
(3ꢁ20 mL). Aqueous layers were extracted with EtOAc
(3ꢁ20 mL) and combined organic layers were dried over
Na2SO4. Filtration and concentration followed by chromato-
graphy on silica gel (2–10% EtOH/CH2Cl2) gave the title
compound 30 (505 mg, 82%, two steps) as a colorless solid.
[a]2D4 ꢂ2.53 (c 2.96, MeOH). IR (Nujol) 1685, 1636, 1540,
A solution of the secondary amine in AcOH (1 mL) was hy-
drogenated over 20% Pd(OH)2 on carbon (20 mg) for 3 h.
The mixture was filtrated through Celite and the filtrate
was evaporated to dryness. The resultant residue was dis-
solved in water and was purified by preparative RP-HPLC
with a linear gradient from 5% H2O/MeCN containing
0.1% TFA to 50% H2O/MeCN containing 0.1% TFA in
20 min. Joramine (7) was eluted at 10.24 min and was ob-
tained as a colorless TFA salt (22 mg, 69%). [a]2D2 ꢂ10.29
1480, 1367, 1164, 854 cmꢂ1
.
1H NMR (300 MHz,
CD3OD): d 1.26 (m, 2H), 1.39 (s, 9H), 1.43 (m, 4H), 1.69
(quin, 2H, J¼7.2 Hz), 2.36 (t, 2H, J¼6.6 Hz), 2.52 (dd,
1H, J¼5.7, 16.2 Hz), 2.59 (dd, 1H, J¼5.7, 16.2 Hz), 2.84
(m, 2H), 3.07 (m, 2H), 3.13 (t, 2H, J¼6.6 Hz), 3.26 (m,
2H), 3.44 (m, 2H), 4.32 (dd, 1H, J¼5.1, 5.7 Hz), 5.05 (s,
2H), 7.28 (m, 5H), 7.75 (m, 3H), 7.99 (m, 1H). 13C NMR
(75 MHz, CD3OD): d 25.1, 28.7, 29.8, 29.9, 35.8, 36.4,
38.4, 40.3, 41.8, 41.9, 45.0, 46.2, 53.1, 63.4, 80.9, 125.9,
128.9, 129.1, 129.6, 131.5, 133.6, 134.8, 135.0, 138.1,
150.0, 157.5, 158.1, 173.5, 173.8, 175.1. FABHRMS calcd
for C34H49N7O11SNa (M++Na) 786.3129, found 786.3119.
(c 0.35, H2O). IR (Nujol) 3479, 2924, 1696, 1515 cmꢂ1
.
1H NMR (300 MHz, D2O): d 1.05 (quin, 2H, J¼6.9 Hz),
1.28 (quin, 4H, J¼6.9 Hz), 1.96 (quin, 2H, J¼8.1 Hz),
2.53 (t, 2H, J¼6.9 Hz), 2.57 (dd, 1H, J¼8.1, 15.3 Hz),
2.64 (dd, 1H, J¼6.0, 15.3 Hz), 3.00 (m, 8H), 3.18 (t, 2H,
J¼6.9 Hz), 3.42 (s, 2H), 4.46 (dd, 1H, J¼6.0, 8.1 Hz),
6.74 (d, 2H, J¼8.1 Hz), 7.05 (d, 2H, J¼8.1 Hz). 13C NMR
(75 MHz, D2O): d 22.8, 23.3, 27.4, 27.5, 30.6, 36.0, 36.2,
38.9, 39.0, 41.0, 43.4, 44.2, 50.7, 115.4, 126.4, 130.2,
154.4, 171.2, 171.9, 174.2, 174.6. FABHRMS calcd for
C23H38N6O5 (M+H)+ 479.3008, found 479.2959. ESI-MS/
MS: Supplementary data 8.
4.1.30. N-[Na-(4-Benzyloxyphenylacetyl)-L-asparaginyl]-
N0-[7-(2-nitrobenzenesulfonylamino)-4-benzyloxycar-
bonyl-4-azaheptanoyl]-1,5-diaminopentane (31). To
a cold (0 ꢀC) and stirred suspension of polyamine 30
(170 mg, 223 mmol) in CHCl3 (1 mL) was added TFA
(1 mL) slowly. After being stirred for 1 h at room tempera-
ture, the reaction mixture was concentrated in vacuo to
give the crude amine TFA salt as a colorless solid.
4.1.32. 7-Allyloxycarbonylamino-4-(2-nitrobenzen-
sulfonyl)-4-azaheptan-1-yl bromide (32). To a solution
of N-(2-nitrobenzenesulfonyl)-N0-(allyloxycarbonyl)-1,3-
diaminopropane (600 mg, 1.75 mmol), which was prepared
from sulfonamide 20 in 93% yield,18 and 1,3-dibromopro-
pane (1.06 g, 5.20 mmol) in DMF (10 mL) was added
Cs2CO3 (855 mg, 2.62 mmol). After being stirred for 0.5 h
at 50 ꢀC, the reaction mixture was diluted with EtOAc
(200 mL) and washed with H2O (3ꢁ40 mL) and brine
(3ꢁ40 mL). Aqueous layers were extracted with EtOAc
(3ꢁ50 mL) and combined organic layers were dried over
Na2SO4. Filtration and concentration followed by chromato-
graphy on silica gel (20–40% EtOAc/hexane) gave the title
To a stirred solution of obtained amine TFA salt in DMF
(1 mL) were added Et3N (23 mg, 228 mmol) and 4-benzyl-
oxyphenylacetic acid succinimide ester31 (114 mg,
336 mmol) slowly. After being stirred for 2 h at room temper-
ature, the reaction mixture was diluted with EtOAc
(100 mL) and washed with saturated NaHCO3 solution
(3ꢁ20 mL) and brine (3ꢁ20 mL). Aqueous layers were ex-
tracted with EtOAc (3ꢁ20 mL) and combined organic layers
were dried over Na2SO4. Filtration and concentration
followed by chromatography on silica gel (2–10% EtOH/

8348
K.-i. Nihei et al. / Tetrahedron 62 (2006) 8335–8350
compound 32 (730 mg, 90%) as a colorless oil. IR (film)
J¼6.3, 15.0 Hz), 2.39 (dd, 1H, J¼6.0, 15.0 Hz), 2.69 (m,
4H), 3.17 (m, 8H), 3.84 (m, 3H), 4.16 (dd, 1H, J¼6.0,
6.3 Hz), 4.97 (s, 2H), 5.02 (s, 2H), 5.20 (s, 2H), 6.81 (d,
1H, J¼7.5 Hz), 6.86 (br s, 1H), 7.32 (m, 17H), 7.67 (br s,
1H), 7.87 (m, 9H), 9.14 (br s, 2H). 13C NMR (75 MHz,
DMSO-d6): d 23.3, 25.3, 26.9, 27.5, 28.3, 28.4, 28.7, 29.3,
36.2, 37.6, 38.6, 44.6, 44.8, 45.0, 45.6, 47.3, 51.7, 54.8,
65.6, 66.3, 68.4, 78.3, 124.5, 124.6, 127.8, 127.9, 128.1,
128.4, 128.5, 128.7, 129.9, 131.8, 131.9, 132.7, 134.7,
134.8, 135.5, 137.2, 137.3, 147.7, 155.2, 155.3, 156.2,
159.9, 163.1, 171.4, 171.8. FABHRMS calcd for
C62H78N12O19S2Na (M+Na)+ 1381.4845, found 1381.4835.
1
1715, 1546, 1440, 1373, 1247, 1162, 931, 852 cmꢂ1. H
NMR (300 MHz, CDCl3): d 1.80 (quin, 2H, J¼6.3 Hz),
2.10 (quin, 2H, J¼6.3 Hz), 3.25 (q, 2H, J¼6.3 Hz), 3.40
(m, 6H), 4.56 (d, 2H, J¼5.4 Hz), 5.08 (br s, 1H), 5.22 (dd,
1H, J¼1.8, 10.8 Hz), 5.31 (dd, 1H, J¼1.8, 15.9 Hz), 5.92
(ddd, 1H, J¼5.4, 10.8, 15.9 Hz), 7.66 (m, 1H), 7.72 (m,
2H), 8.04 (m, 1H). 13C NMR (75 MHz, CDCl3): d 28.1,
29.7, 31.1, 37.5, 45.3, 45.6, 65.4, 117.5, 124.2, 130.9,
131.6, 132.7, 133.6, 147.9, 156.2. HRMS calcd for
C10H18N2O2Br (M+ꢂNs) 277.0551, found 277.0564.
4.1.33. N-(Na-tert-Butoxycarbonyl-L-asparaginyl)-12-
(Na,Nd,Nu-tribenzyloxycarbonylarginyl)-amino-6,10-
[di-(2-nitrobenzenesulfonyl)]-6,10-diaza-1-aminounde-
cane (34). To a solution of sulfonamide 10 (200 mg,
399 mmol) and bromide 32 (278 mg, 599 mmol) in DMF
(2 mL) were added Cs2CO3 (391 mg, 1.20 mmol) and
TBAI (15.0 mg, 40.6 mmol). After being stirred for 1 h at
70 ꢀC, the reaction mixture was diluted with EtOAc
(150 mL) and washed with H2O (3ꢁ30 mL) and brine
(3ꢁ30 mL). Aqueous layers were extracted with EtOAc
(3ꢁ30 mL) and combined organic layers were dried over
Na2SO4. Filtration and concentration followed by chromato-
graphy on silica gel (2–4% MeOH/CH2Cl2) gave the title
compound 33 (332 mg), which was used in the next step
without further purification. 1H NMR (300 MHz,
CD3COCD3): d 1.27 (m, 2H), 1.40 (s, 9H), 1.48 (m, 4H),
1.78 (quin, 2H, J¼6.9 Hz), 1.86 (quin, 2H, J¼7.2 Hz),
2.59 (dd, 1H, J¼6.6, 15.9 Hz), 2.73 (dd, 1H, J¼4.8,
15.9 Hz), 3.16 (m, 4H), 3.36 (m, 8H), 4.35 (dd, 1H, J¼4.8,
6.6 Hz), 4.50 (d, 2H, J¼5.4 Hz), 5.14 (dd, 1H, J¼1.2,
10.8 Hz), 5.27 (dd, 1H, J¼1.2, 16.2 Hz), 5.92 (ddd, 1H,
J¼5.4, 10.8, 16.2 Hz), 6.36 (m, 3H), 6.98 (br s, 1H), 7.33
(br s, 1H), 7.87 (m, 6H), 8.06 (m, 2H). 13C NMR
(75 MHz, CD3COCD3): d 24.6, 28.3, 28.8, 28.9, 30.1,
38.4, 39.3, 39.8, 46.1, 46.3, 46.7, 48.8, 52.7, 65.8, 79.9,
117.5, 125.5, 125.6, 131.5, 133.3, 133.4, 135.1, 135.4,
135.5, 149.5, 156.6, 157.4, 172.3, 173.9.
4.1.34. N-(Na-2,4-Dibenzyloxyphenylacetyl-L-aspara-
ginyl)-12-(Na,Nd,Nu-tribenzyloxycarbonylarginyl)-amino-
6,10-[di-(2-nitrobenzenesulfonyl)]-6,10-diaza-1-amino-
undecane (35). To a cold (0 ꢀC) and stirred suspension of
polyamine 34 (90 mg, 66.2 mmol) in CHCl3 (1 mL) was added
TFA (1 mL) slowly. After being stirred for 1 h at room tem-
perature, the reaction mixture was concentrated in vacuo to
give the crude amine TFA salt as a colorless solid.
To a stirred solution of obtained amine TFA salt in DMF
(1 mL) were added Et3N (7 mg, 69.2 mmol) and 2,4-di-
benzyloxyphenylacetic acid succinimide ester28 (45 mg,
101 mmol) slowly. After being stirred for 2 h at room temper-
ature, the reaction mixture was directly subjected to silica
gel chromatography (2–10% MeOH/CH2Cl2) to afford the
title compound 35 (84 mg, 80%, two steps) as a white
powder. [a]2D4 ꢂ0.43 (c 0.70, DMSO). IR (Nujol) 1721,
1
1658, 1545, 1457, 1377, 1262, 1162, 852 cmꢂ1. H NMR
(300 MHz, DMSO-d6): d 1.06 (m, 2H), 1.23 (m, 2H), 1.33
(m, 2H), 1.54 (m, 8H), 2.35 (dd, 1H, J¼7.2, 15.0 Hz), 2.42
(dd, 1H, J¼6.3, 15.0 Hz), 2.89 (m, 2H), 2.97 (m, 2H), 3.16
(m, 8H), 3.37 (m, 2H), 3.83 (m, 3H), 4.51 (m, 1H), 4.96 (s,
2H), 5.01 (s, 2H), 5.02 (s, 2H), 5.07 (s, 2H), 5.18 (s, 2H),
6.51 (s, 2H), 6.67 (br s, 1H), 6.85 (br s, 1H), 7.07 (m, 2H),
7.33 (m, 26H), 7.61 (m, 1H), 7.85 (m, 10H), 9.14 (br
s, 2H). 13C NMR (75 MHz, DMSO-d6): d 23.4, 25.3, 26.8,
27.5, 28.2, 28.7, 29.3, 36.2, 36.4, 37.5, 38.7, 44.6, 44.7,
45.0, 45.6, 47.3, 50.0, 54.8, 65.6, 66.3, 68.4, 69.4, 69.5,
127.3, 127.8, 127.9, 128.0, 128.1, 128.5, 128.6, 128.7, 129.9,
131.2, 131.8, 131.9, 132.7, 134.7, 134.8, 135.5, 137.2, 137.3,
137.4, 147.7, 155.2, 156.2, 157.1, 158.5, 160.0, 163.1, 170.5,
170.9, 171.7, 171.8. FABHRMS calcd for C79H88N12O20S2Na
(M+Na)+ 1611.5577, found 1611.5571.
To a cold (0 ꢀC) and stirred solution of polyamine 33
(135 mg, 153 mmol) in CH2Cl2 (4 mL) were added PPh3
(8 mg, 30.5 mmol), Pd(PPh3)4 (9 mg, 7.8 mmol), and pyrroli-
dine (54 mg, 759 mmol). After being stirred for 0.5 h at room
temperature, the reaction mixture was concentrated in vacuo.
The residue was subjected to silica gel chromatography (2–
10% EtOH/CH2Cl2, then 7.5% MeOH/CHCl3 containing
2.5% i-PrNH2) to afford the primary amine as a colorless oil.
4.1.35. Argiotoxin-636 (8). To a stirred solution of poly-
amine 35 (40 mg, 25.2 mmol) in DMF (1 mL) were added
2-mercaptoethanol (39 mg, 499 mmol) and DBU (77 mg,
506 mmol). After being stirred for 0.5 h at room temperature,
the reaction mixture was directly purified on silica gel
column (2–12% EtOH/CH2Cl2, then, 7.5% MeOH/CHCl3
containing 2.5% i-PrNH2) and then, further purification
by preparative TLC (7.5% MeOH/CHCl3 containing 2.5%
i-PrNH2) gave the secondary amine as a white powder.
To a stirred solution of amine obtained in DMF (1 mL) was
added tri-Cbz-arginine succinimide ester48 (154 mg,
229 mmol) slowly. After being stirred for 1 h at room temper-
ature, the reaction mixture was diluted with EtOAc
(100 mL) and washed with saturated NaHCO3 solution
(3ꢁ20 mL) and brine (3ꢁ20 mL). Aqueous layers were ex-
tracted with EtOAc (3ꢁ20 mL) and combined organic layers
were dried over Na2SO4. Filtration and concentration
followed by chromatography on silica gel (2–6% MeOH/
CH2Cl2) gave the title compound 34 (146 mg, 66%, three
steps from 10) as a white powder. [a]2D2 +3.06 (c 1.11,
DMSO). IR (Nujol) 1727, 1681, 1646, 1544, 1456, 1376,
A solution of the secondary amine in AcOH (1 mL) was hy-
drogenated over 20% Pd(OH)2 on carbon (20 mg) for 2 h.
The mixture was filtrated through Celite and the filtrate
was evaporated to dryness. The resultant residue was dis-
solved in water and was purified by preparative RP-HPLC
with a linear gradient from 5% H2O/MeCN containing
1
1255, 1162, 852 cmꢂ1. H NMR (300 MHz, DMSO-d6):
d 1.09 (m, 2H), 1.34 (m, 13H), 1.55 (m, 8H), 2.32 (dd, 1H,

K.-i. Nihei et al. / Tetrahedron 62 (2006) 8335–8350
8349
15. Miyashita, M.; Kanemura, T.; Matsushita, M.; Hatakeyama, S.;
Itagaki, Y.; Nakajima, T.; Miyazawa, M.; Irie, H. Heterocycles
1988, 47, 171.
16. Wakamiya, T.; Yamamoto, A.; Kawaguchi, K.; Kinoshita, T.;
Yamaguchi, Y.; Itagaki, Y.; Naoki, H.; Nakajima, T. Bull.
Chem. Soc. Jpn. 2001, 74, 1743.
17. A preliminary account of this work: Nihei, K.; Kato, M. J.;
Yamane, T.; Palma, M. S.; Konno, K. Bioorg. Med. Chem.
Lett. 2002, 12, 299.
18. Fukuyama, T.; Jow, C.-K.; Cheung, M. Tetrahedron Lett. 1995,
36, 6373.
19. Fukuyama, T.; Cheung, M.; Jow, C. K.; Hidai, Y.; Kan, T.
Tetrahedron Lett. 1997, 38, 5831.
20. Kan, T.; Fukuyama, T. Chem. Commun. 2004, 353.
21. Hidai, Y.; Kan, T.; Fukuyama, T. Tetrahedron Lett. 1999, 40,
4711.
0.1% TFA to 50% H2O/MeCN containing 0.1% TFA in
20 min. Arg-636 (8) was eluted at 9.06 min and was ob-
tained as a colorless TFA salt (14 mg, 51%, two steps).
[a]2D2 +4.69 (c 0.98, H2O). IR (Nujol) 3394, 2923,
1
1674 cmꢂ1. H NMR (300 MHz, D2O): d 1.07 (quin, 2H,
J¼7.2 Hz), 1.32 (m, 2H), 1.45 (quin, 2H, J¼7.2 Hz), 1.50
(quin, 2H, J¼7.2 Hz), 1.77 (m, 4H), 1.95 (m, 2H), 2.56
(dd, 1H, J¼6.9, 15.0 Hz), 2.62 (dd, 1H, J¼6.9, 15.0 Hz),
2.95 (m, 8H), 3.10 (m, 6H), 3.33 (d, 1H, J¼15.6 Hz), 3.43
(d, 1H, J¼15.6 Hz), 3.83 (t, 1H, J¼7.2 Hz), 4.44 (t, 1H,
J¼6.9 Hz), 6.30 (m, 2H), 6.95 (d, 1H, J¼8.7 Hz). 13C
NMR (75 MHz, D2O): d 22.5, 22.7, 23.5, 24.9, 25.3, 27.5,
27.9, 35.8, 36.4, 36.8, 38.8, 40.2, 44.1, 44.4, 45.2, 47.5,
50.9, 52.9, 102.7, 107.4, 113.7, 132.4, 155.1, 156.1, 156.7,
169.6, 172.4, 174.6, 174.9. FABHRMS calcd for
C29H53N10O6 (M++H) 665.4162, found 665.4127. ESI-
MS/MS: Supplementary data 9.
22. Hidai, Y.; Kan, T.; Fukuyama, T. Chem. Pharm. Bull. 2000, 48,
1570.
23. Kan, T.; Kobayashi H.; Fukuyama, T. Synlett 2002, 1338.
24. Aramaki, Y.; Yasuhara, T.; Higashijima, T.; Yoshioka, M.;
Miwa, A.; Kawai, N.; Nakajima, T. Proc. Jpn Acad. 1986,
62B, 359.
25. The free amine of mono-Ns-cadaverine is not stable enough,
gradually decomposes during stored, but its HCl salt is quite
stable.
26. Nihei, K.; Kato, M. J.; Yamane, T.; Palma, M. S.; Konno, K.
Synlett 2001, 1167.
27. Yamamoto, H.; Maruoka, K. J. Am. Chem. Soc. 1981, 103,
6133.
Acknowledgements
We are grateful to Professors Toshiyuki Kan (University of
Shizuoka), Shojiro Maki (The University of Electro-Com-
munication), and Yoshitomo Suhara (Kobe Pharmaceutical
University) for their invaluable discussions and technical
assistance. Thanks are also due to Dr. Hideo Naoki (Okinawa
Health Biotechnology Research Development Center) for
the measurement of the HRMS spectra.
28. Matsushita, M.; Kanemura, T.; Hatakeyama, S.; Irie, H.; Toki,
T.; Miyashita, M. Tetrahedron 1995, 51, 10687.
29. Toki, T.; Yasuhara, T.; Aramaki, Y.; Kawai, N.; Nakajima, T.
Biomed. Res. 1988, 9, 75.
30. Toki, T.; Yasuhara, T.; Aramaki, Y.; Osawa, K.; Miwa, A.;
Kawai, N.; Nakajima, T. Biomed. Res. 1988, 9, 421.
31. Shinada, T.; Miyachi, M.; Itagaki, Y.; Naoki, H.; Yoshihara, K.;
Nakajima, T. Tetrahedron Lett. 1996, 37, 7099.
32. Palma, M. S.; Itagaki, Y.; Fujita, T.; Naoki, H.; Nakajima, T.
Toxicon 1998, 36, 485.
33. McCormick, K. D.; Kobayashi, K.; Goldin, S. M.; Reddy,
N. L.; Meinwald, J. Tetrahedron 1993, 49, 11155.
34. Miyashita, M.; Matsushita, M.; Sato, H.; Toki, T.; Nakajima,
T.; Irie, H. Chem. Lett. 1993, 929.
35. Miyashita, M.; Saito, H.; Matsushita, M.; Miyazawa, M.;
Itagaki, Y.; Nakajima, T. Tetrahedron Lett. 1997, 38, 8297.
36. Eldefrawi, A. T.; Eldefrawi, M. E.; Konno, K.; Mansour, N. A.;
Nakanishi, K.; Oltz, E.; Usherwood, P. N. R. Proc. Natl. Acad.
Sci. U.S.A. 1988, 85, 4910.
37. Piek, T.; Hue, B. Comp. Biochem. Physiol., C 1989, 93, 403.
38. Strømgaard, K.; Andersen, K.; Krogsgaard-Larsen, P.;
Jaroszewski, J. W. Mini Rev. Med. Chem. 2001, 1, 317.
39. Teshima, T.; Wakamiya, T.; Aramaki, Y.; Nakajima, T.; Kawai,
N.; Shiba, T. Tetrahedron Lett. 1987, 28, 3509.
40. Nason, D. M.; Jasys, V. J.; Kelbaugh, P. R.; Phillips, D.;
Saccomano, N. A.; Volkman, R. Tetrahedron Lett. 1989, 30,
2337.
41. Blagbrough, I. S.; Moya, E.; Walford, S. P. Tetrahedron Lett.
1996, 37, 551.
42. Chiba, T.; Akizawa, T.; Matsukawa, M.; Pan, H. H.; Yoshioka,
M. Chem. Pharm. Bull. 1994, 42, 1864.
Supplementary data
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.tet.2006.06.051.
References and notes
1. McCormick, K. D.; Meinwald, J. J. Chem. Ecol. 1993, 19,
2411.
2. Schulz, S. Angew. Chem., Int. Ed. 1997, 36, 314.
3. Mellor, I. R.; Usherwood, P. N. R. Toxicon 2004, 43, 493.
4. Albensi, B. C.; Ilkanich, E. Drug News Perspect. 2004, 17, 557.
5. Saito, H.; Yuri, E.; Miyazawa, M.; Itagaki, Y.; Nakajima, T.;
Miyashita, M. Tetrahedron Lett. 1998, 39, 6479.
6. Hashimoto, M.; Liu, Y.; Fang, K.; Li, H.; Campiani, G.;
Nakanishi, K. Bioorg. Med. Chem. 1999, 7, 1181.
7. Wang, F.; Manku, S.; Hall, D. G. Org. Lett. 2000, 2, 1581.
8. Shinada, T.; Nakagawa, Y.; Hayashi, K.; Corzo, G.; Nakajima,
T.; Ohfune, Y. Amino Acids 2003, 24, 293.
9. Fujita, T.; Itagaki, Y.; Hisada, M.; Naoki, H.; Nakajima, T.
Rapid Commun. Mass Spectrom. 1995, 9, 365.
10. Fujita, T.; Itagaki, Y.; Hisada, M.; Naoki, H.; Nakajima, T.;
Andriantsiferana, M. Rapid Commun. Mass Spectrom. 1997,
11, 1115.
11. Itagaki, Y.; Fujita, T.; Naoki, H.; Yasuhara, T.;
Andriantsiferana, M.; Nakajima, T. Nat. Toxins 1997, 5, 1.
12. Hisada, M.; Fujita, T.; Naoki, H.; Itagaki, Y.; Irie, H.;
Miyashita, M.; Nakajima, T. Toxicon 1998, 36, 1115.
13. Itagaki, Y.; Nakajima, T. J. Toxicol. Toxin Rev. 2000, 19, 23.
14. Wakamiya, T.; Kinoshita, T.; Hattori, Y.; Yamaguchi, Y.;
Naoki, H.; Corzo, G.; Nakajima, T. Bull. Chem. Soc. Jpn.
2004, 77, 331.
43. Budd, T.; Clinton, P.; Dell, A.; Duce, I. R.; Johnson, S. J.;
Quicke, D. L. J.; Taylor, G. W.; Usherwood, P. N. R.; Usoh,
G. Brain Res. 1988, 448, 30.

8350
K.-i. Nihei et al. / Tetrahedron 62 (2006) 8335–8350
44. Grishin, E. V.; Volkova, T. M.; Arseniev, A. S. Toxicon 1989,
27, 541.
50. Raditsch, M.; Geyger, M.; Kalbitzer, H. R.; Jahn, W.;
Ruppersberg, J. P.; Witzemann, V. Eur. J. Biochem. 1996,
240, 416.
45. Grishin, E. V.; Volkova, T. M.; Arseniev, A. S.; Reshetova,
O. S.; Onoprienko, V. V.; Magazanic, L. G.; Anotonov,
S. M.; Fedorova, I. M. Bioorg. Khim. 1986, 12, 1121.
46. Deziel, R. Tetrahedron Lett. 1987, 28, 4371.
47. Shih, T. L.; Ruiz-Sanchez, J.; Mrozik, H. Tetrahedron Lett.
1987, 28, 6015.
48. Jasys, J. V.; Kelbaugh, P. R.; Nason, D. M.; Phillips, D.;
Saccomano, N. A.; Volkmann, R. A. Tetrahedron Lett. 1988,
29, 6222.
51. Salamoni, S. D.; Costa, J. C.; Palma, M. S.; Konno, K.; Nihei,
K.; Tavares, A. A.; Abreu, D. S.; Venturin, G. T.; Cunha, F. B.;
Oliveira, R. M.; Breda, R. V. Brain Res. 2005, 1048, 170.
52. Salamoni, S. D.; Costa, J. C.; Palma, M. S.; Konno, K.; Nihei,
K.; Azambuja, N. A.; Neto, E. P.; Venturin, G. T.; Tavares,
A. A.; Abreu, D. S.; Breda, R. V. NeuroReport 2005, 16, 1869.
53. Chiba, T.; Akizawa, T.; Matsukawa, M.; Nishi, M.; Kawai, N.;
Yoshioka, M. Chem. Pharm. Bull. 1996, 44, 972.
49. Blagbrough, I. S.; Moya, E. Tetrahedron Lett. 1995, 36, 9393.
54. Pfeiffer, M. J.; Samir, B. H. J. Org. Chem. 1993, 58, 735.