Study on the Structure Activity Relationships of NPTX-594, a Spider Toxin Belonging to the Type-B Acylpolyamine Structure

Article abstract of DOI:10.1246/bcsj.77.331

In order to elucidate the structure activity relationships of the spider toxin termed NPTX-594, eleven toxin analogs were designed and synthesized, and their paralytic activities against cricket were tested. As a result of the present study, it was clarif

Full text of DOI:10.1246/bcsj.77.331

ꢀ 2004 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 77, 331–340 (2004)
331
Study on the Structure Activity Relationships of NPTX-594,
a Spider Toxin Belonging to the Type-B Acylpolyamine Structure#
ꢀ
Tateaki Wakamiya, Tomohiko Kinoshita, Yoshihide Hattori, Yoshihiro Yamaguchi,
Hideo Naoki,1 Gerardo Corzo,1 and Terumi Nakajima1
Faculty of Science and Technology, Kinki University, Higashi-osaka, Osaka 577-8502
1Suntory Institute for Bioorganic Research, Shimamoto-cho, Mishima-gun, Osaka 618-8503
Received August 1, 2003; E-mail: wakamiya@chem.kindai.ac.jp
In order to elucidate the structure activity relationships of the spider toxin termed NPTX-594, eleven toxin analogs
were designed and synthesized, and their paralytic activities against cricket were tested. As a result of the present study, it
was clarified that the Lys residue binding to the 1-amino group of 4,8-diaza-1,12-dodecanediamine (Dada) in the molecule
of NPTX-594 is not an essential requisite for toxicity, and can be replaced with neutral or basic amino acids without any
considerable loss of the activity. However, the replacement of the Lys residue with acidic amino acid residues, such as
Asp or Glu, resulted in an extreme loss of the biological activity.
Spider venom contains many structural types of acylpoly-
amine toxins1–15 that are known to be potent and specific block-
ers against the glutamate receptor.16 We recently synthesized a
novel spider toxin termed NPTX-594 (1) (Fig. 1) isolated from
Nephila madagascariencis, a Madagascar Joro spider, or from
Nephila clavipes, a Brazilian Joro spider.17 NPTX-594 is com-
prised of four residues, i.e., 2,4-dihydroxyphenylacetic acid
(Dhpa), Asn, 4,8-diaza-1,12-dodecanediamine (Dada), and
Lys.18 Acylpolyamine spider toxins found in nature are classi-
fied into six structural types.15 NPTX-594 containing Dada as
the polyamine component belongs to the structural type-B. In
order to elucidate the structure–activity relationships of this
novel toxin, we focused on the role of the Lys residue, which
binds to the 1-amino group of Dada, to exhibit biological activ-
ity. For this purpose, eleven NPTX-594 analogs, such as 2–12
(Fig. 2), were designed and synthesized, i.e., the Lys residue
was deleted or replaced with several basic, neutral, or acidic
amino acid residues.
(Abg = 7-amino-3-azaheptanoic acid), and Arg residues at
the place of the Lys residue. For this purpose, three unusual ba-
sic amino acids, i.e., Pua, Apa, and Abg, were prepared, as
shown in Schemes 1–3.
Schemes 1 and 2 show the routes to prepare Pua and Apa
based on the Michael reaction between methyl acrylate and
amino alcohols 13 and 19 to construct the backbone structures,
14 and 20, of the corresponding diamino acids.19,20 The hydoxy
groups of both compounds were converted into the amino
group via the azide compounds, 15 and 21, respectively. These
diamino acids were finally prepared as the active esters, 18 and
24, for the coupling with 33 that is the segment corresponding
to des-Lys-(NPTX-594). The Abg derivative was prepared by
employing the conventional reductive N-alkylation method be-
tween monoprotected diamine 2721,22 and glyoxylic acid, as
shown in Scheme 3; this amino acid was also converted to
the active ester 29.
The synthetic routes of NPTX-594 analogs 2–12 are shown
in Scheme 4. The Boc group in the molecule of 30,17 which
is a fully protected Dada residue, as the key component for
the synthesis of all analogs, was removed by a conventional
method. The Asn and Dhpa20,23,24 residues were then succes-
sively introduced to the freed amino terminal side of the Dada
residue by means of the active ester method, and the compound
32 was obtained as the common intermediate for the synthesis
of all analogs.
Results and Discussion
In acylpolyamine spider toxins, the putreanine [Pua = N-(4-
aminobutyl)-ꢀ-alanine or 8-amino-4-azaoctanoic acid] or the
Arg residue instead of the Lys residue generally binds to one
side of the amino groups in polyamine unit.19 In the present
study we first designed four analogs with the Pua, two Lys
equivalents such as N-(3-aminopropyl)-ꢀ-alanine (Apa = 7-
amino-4-azaheptanoic acid) and N-(4-aminobutyl)glycine
Des-Lys-(NPTX-594) (2) was first obtained from 32 by suc-
Fig. 1. The structure of NPTX-594.
Published on the web February 10, 2004; DOI 10.1246/bcsj.77.331

332 Bull. Chem. Soc. Jpn., 77, No. 2 (2004)
Structure Activity Relationships of NPTX-594
Table 1. Biological Activities (Cricket Bioassay) of NPTX-
594 and Its Analogs
Compounds
NPTX-594
ED50/nmol gꢁ1a)
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
0:64 ꢂ 0:50
3:73 ꢂ 3:32
1:74 ꢂ 1:56
2:47 ꢂ 2:47
0:20 ꢂ 0:22
2:02 ꢂ 1:65
5:24 ꢂ 3:52
4:87 ꢂ 9:51
3:72 ꢂ 1:34
4:53 ꢂ 2:28
67:4 ꢂ 47:8
110 ꢂ 100
Des-Lys-(NPTX-594)
Pua-[Des-Lys-(NPTX-594)]
Apa-[Des-Lys-(NPTX-594)]
Abg-[Des-Lys-(NPTX-594)]
Arg-[Des-Lys-(NPTX-594)]
Acp-[Des-Lys-(NPTX-594)]
Gly-[Des-Lys-(NPTX-594)]
Ser-[Des-Lys-(NPTX-594)]
Phe-[Des-Lys-(NPTX-594)]
Glu-[Des-Lys-(NPTX-594)]
Asp-[Des-Lys-(NPTX-594)]
(9)
(10)
(11)
(12)
a) Crickets (Grillus bimaculatus) were injected intrathoracical-
ly between the second and third pair of legs, with 5 mL of five
different doses of each toxin previously dissolved in Milli-Q
water (18.2 Mꢀ cm). The ED50 value for the toxins is given
in nanomole (nmol) of toxin per g of cricket, which represents
the effective dose to paralyze 50% of treated crickets at 5 min
after injection. The inability of crickets to upturn when they
were placed on their back was employed as the criterion for pa-
ralysis. The ED50 value was obtained by probit analysis of data
from three groups of 5 crickets.
crickets (Grillus bimaculatus). The ED50 values of these ana-
logs are given in Table 1. So far as we examined, the analogs
in which the Lys residue is replaced with the basic amino acid
residues show comparable biological activities to NPTX-594.26
On the other hand, replacing the Lys residue with the acidic
amino acid residues resulted in an extreme loss of biological ac-
tivity. As a result of the present study, it is possible to suggest
that the Lys residue in NPTX-594 is not an essential requisite
for toxicity. In particular, it is noteworthy that the Lys residue
is replaceable with neutral amino acid residues.27 This fact
seems to be quite valuable from the standpoint of the design
of fluorescence-labeled analogs or analogs for photoaffinity-la-
beling to elucidate the mode of action of NPTX-594 as a block-
er against the glutamate receptor.
Fig. 2. NPTX-594 analogs designed and synthesized in the
present study.
cessive removal of the Troc and the benzyl-type protecting
groups. Other analogs 3–12 were prepared by the coupling of
the corresponding N-Z-amino acid succinimidyl esters25 with
33 obtained from the intermediate 32. All of the protecting
groups in the coupling products 41–50 were removed by cata-
lytic hydrogenation in acidic medium, and purified by prepara-
tive RPHPLC.
During the course of the present study, we realized that ana-
logs 2, 3, and 6 might be the same compounds as NPTX-466,
NPTX-608, and NPTX-622 found in the venom of several spi-
ders, respectively. A detailed comparison of these synthetic an-
alogs with natural compounds by mass spectrometry is current-
The biological activities of synthetic acylpolyamines 2–12
obtained as mentioned above were evaluated by paralysis to
Scheme 1. Preparation of the active ester derivative of Pua.

T. Wakamiya et al.
Bull. Chem. Soc. Jpn., 77, No. 2 (2004)
333
Scheme 2. Preparation of the active ester derivative of Apa.
Scheme 3. Preparation of the active ester derivative of Abg.
od. The mixture was stirred for 2.5 h at r. t., and the insoluble ma-
terial was filtered off. The filtrate was concentrated in vacuo, and
the residue was dissolved in AcOEt (50 mL). The solution was
washed with H2O (10 mL), 10% citric acid (10 mL ꢃ 3), and satu-
rated aqueous NaHCO3 (10 mL ꢃ 3). The organic layer was dried
over anhydrous MgSO4, and then concentrated in vacuo to give
methyl 4-benzyloxycarbonyl-8-hydroxy-4-azaoctanoate (14)
(3.00 g, 86.5%) as colorless oil; the product was used for the next
reaction without further purification.
To a solution of the thus-obtained 14 (2.00 g, 6.46 mmol) in pyr-
idine (20 mL) was added MsCl (0.747 g, 7.11 mmol) at 0 ꢄC, and
the mixture was stirred for 1 h at 0 ꢄC. After the addition of diethyl
ether (50 mL), the reaction mixture was washed with saturated
aqueous CuSO4 (20 mL ꢃ 3), saturated aqueous NaHCO3 (10
mL ꢃ 3), and brine (10 mL ꢃ 3). The organic layer was dried over
anhydrous MgSO4, and then concentrated in vacuo. To a solution
of the residue in DMF (40 mL) was added NaN3 (2.52 g, 38.8
mmol) at 80 ꢄC, and the solution was stirred for 1 h at 80 ꢄC. After
the addition of AcOEt (100 mL), the reaction mixture was washed
with 10% citric acid (10 mL ꢃ 3), saturated aqueous NaHCO3 (10
mL ꢃ 3), and brine (10 mL ꢃ 3). The organic layer was dried over
anhydrous MgSO4, and then concentrated in vacuo. The thus-ob-
tained crude product was purified by silica-gel column chromatog-
raphy (Art. 9385, 80 g, 2 ꢃ 50 cm, benzene:AcOEt = 20:1). The
fractions containing the desired product were combined, and con-
centrated in vacuo to give 15 as colorless oil (1.33 g, 61.6%).
1H NMR (CDCl3) ꢁ 1.59 (4H, m, C6H2, C7H2), 2.56 and 2.63 (each
1H, m, C2H2), 3.50 (4H, m, C3H2, C5H2), 3.54 (2H, t, C8H2), 3.65
(3H, s, CH3), 5.12 (2H, s, CH2/Z), 7.28–7.40 (5H, m, Ph/Z).
MALDI-TOF MS: found m=z 335.2 (M + H)þ (calcd for
ly being undertaken, and the results will be reported soon else-
where.
Experimental
All of the melting points are uncorrected, and were measured by
a Yanaco MP-J3 (Yanaco Co., Ltd., Kyoto, Japan). Silica-gel col-
umn chromatography was carried out with Merck silica gel 60 (Art.
7734, 70–230 mesh) or with Merck silica gel 60 (Art. 9385, 230–
400 mesh) at medium pressure (1–5 kg cmꢁ2). 1H NMR spectra
were recorded on a Mercury 300 (300 MHz, Varian Co., Ltd.,
Germany) or a DMX-500 (500 MHz, Bruker Co., Ltd., Germany).
MALDI TOF-MS was carried out with a Kratos Kompact MALDI
4 (Shimadzu Co., Ltd., Kyoto, Japan). HRFAB-MS was carried out
with a HX110 (JEOL Co., Ltd., Tokyo, Japan). RPHPLC was
carried out with a Shimadzu SCL-10A VP (Shimadzu Co., Ltd.,
Kyoto, Japan), and performed on a Cosmosil 5C18-AR (4:6 ꢃ
150 mm, Nacalai Tesque Co., Ltd., Kyoto, Japan) for analysis
and a YMC-Pack ODS-AM (20 ꢃ 250 mm, YMC Co Ltd, Kyoto,
Japan) for preparative purification. Boc-Asn-ONp was purchased
from Watanabe Chemical Industries Co., Ltd., (Hiroshima Japan).
Solid H2S was purchased from Wako Pure Chemical Industries
Co., Ltd., (Osaka, Japan). Celite 545@ was purchased from Nacalai
Tesque Co., Ltd., (Kyoto, Japan).
Methyl 8-Azido-4-benzyloxycarbonyl-4-azaoctanoate (15).
After methyl acrylate (1.15 g, 13.4 mmol) was added to 4-ami-
no-1-butanolꢄ (13) (1.00 g, 11.2 mmol), the mixture was stirred
for 1 h at 0 C and additionally for 29 h at r. t. To a solution of
the thus-obtained methyl 8-hydroxy-4-azaoctanoate and TEA
(1.58 g, 15.7 mmol) in THF (10 mL) was added dropwise ZCl
(2.29 g, 13.4 mmol) in THF (10 mL) at r. t. over a 30-minute peri-

334 Bull. Chem. Soc. Jpn., 77, No. 2 (2004)
Structure Activity Relationships of NPTX-594
Scheme 4. Synthetic routes to NPTX-594 analogs.
ꢄ
C16H22N4O4 + H: 335.2), 357.2 (M + Na)þ (calcd for
C16H22N4O4 + Na: 357.2).
60 C. The reaction mixture was neutralized with 1 M HCl, and
MeOH was removed in vacuo. The aqueous solution was extracted
with CHCl3 (30 mL ꢃ 3), and the extract was washed with brine
(20 mL ꢃ 3). The organic layer was dried over anhydrous MgSO4,
and then concentrated in vacuo to give colorless oil (0.851 g,
87.9%); the crude product was pure enough to use for the next re-
Methyl
4,N8-Bis(benzyloxycarbonyl)-8-amino-4-azaocta-
noate (16). To a solution of 15 (1.00 g, 2.99 mmol) in THF (10
mL) and H2O (1 mL) was added PPh3 (0.942 g, 3.59 mmol) at 0
ꢄC. The reaction mixture was stirred for 1 h at 0 ꢄC and addition-
ally for 20 h at r. t., and concentrated in vacuo. The residue was
dissolved in AcOEt (50 mL), and it was extracted with 1 M HCl
(10 mL ꢃ 3). The acidic aqueous extract was neutralized with
Na2CO3, and diluted with THF (20 mL). To a mixture basified
by the addition of Na2CO3 (0.634 g, 5.98 mmol) was added drop-
wise ZCl (0.765 g, 5.98 mmol). The reaction mixture was stirred for
5 h at r. t., and then extracted with AcOEt (30 mL ꢃ 3). The extract
was dried over anhydrous MgSO4, and concentated in vacuo. The
crude product was purified by silica-gel column chromatography
(Art. 7734, 38 g, 1 ꢃ 40 cm, CHCl3:MeOH = 20:1). The fractions
containing the desired product were combined, and concentrated in
vacuo to give 16 as colorless oil (1.05 g, 79.5%). 1H NMR (CDCl3)
ꢁ 1.53 (4H, m, C6H2, C7H2), 2.58 (2H, t, C2H2), 3.16–3.28 (4H, m,
C3H2, C5H2), 3.52 (2H, t, C8H2), 3.65 (3H, s, CH3), 5.09 (2H, s,
CH2/Z), 5.12 (2H, s, CH2/Z), 7.31–7.40 (10H, m, Ph/Z). MALDI
TOF-MS: found m=z 443.5 (M + H)þ (calcd for C24H30N2O6 + H:
443.2), 465.4 (M + Na)þ (calcd for C24H30N2O6 + Na: 465.2).
4,N8-Bis(benzyloxycarbonyl)-8-amino-4-azaoctanoic Acid
[Z-Pua(Z)-OH] (17). Methyl 4,N8-bis(benzyloxycarbonyl)-8-
amino-4-azaoctanoate (16) (1.00 g, 2.26 mmol) was dissolved in
1 M NaOH (15 mL) and MeOH (15 mL), and stirred for 1 h at
action. H NMR (CDCl3) ꢁ 1.52 (4H, m, C6H2, C7H2), 2.58 (2H,
1
m, C2H2), 3.15 (2H, m, C3H2) 3.28 (2H, m, C5H2), 3.52 (2H, t,
C8H2), 5.09 (2H, s, CH2/Z), 5.12 (2H, s, CH2/Z), 7.27–7.37
(10H, m, Ph/Z). MALDI TOF-MS: found m=z 451.2 (M + Na)þ
(calcd for C23H28N2O6 + Na: 451.2).
4,N8-Bis(benzyloxycarbonyl)-8-amino-4-azaoctanoic Acid
Succinimidyl Ester [Z-Pua(Z)-ONSu] (18). To a solution of
17 (0.300 g, 0.700 mmol) in DMF (15 mL) were added HONSu
(0.148 g, 12.5 mmol) and EDC HCl (0.0763 g, 0.770 mmol),
ꢅ
and the mixture was stirred overnight at r. t. The reaction mixture
was concentrated in vacuo, and the residue was dissolved in AcOEt
(30 mL). The solution was washed with 10% citric acid (5 mL ꢃ
3), saturated aqueous NaHCO3 (5 mL ꢃ 3), and brine (5 mL ꢃ
3). The organic layer was dried over anhydrous MgSO4, and then
concentrated in vacuo to give 18 (0.295 g, 80.2%) as colorless oil.
The thus-obtained crude product was used for the next reaction
without further purification.
Methyl 7-Azido-4-benzyloxycarbonyl-4-azaheptanoate (21).
Methyl acrylate (3.78 g, 43.9 mmol) was added to 3-amino-1-prop-
anol (19) (3.00 g, 39.9 mmol), and the mixture was stirred for 1 h at
0 ꢄC and additionally for 32 h at r. t. To a solution of the thus-ob-

T. Wakamiya et al.
Bull. Chem. Soc. Jpn., 77, No. 2 (2004)
335
tained methyl 7-hydroxy-4-azaheptanoate in THF (100 mL) was
added TEA (4.50 g, 44.5 mmol), and was then added dropwise
ZCl (8.17 g, 47.9 mmol) in THF (50 mL) at r. t. over a 30-minute
period. The mixture was stirred overnight at r. t., and worked up in
a similar manner as described in the preparation of 14. The thus-
obtained crude product was purified by silica-gel column chroma-
tography (Art. 7734, 180 g, 2 ꢃ 70 cm, CHCl3:MeOH = 50:1).
The fractions containing the desired product were combined, and
concentrated in vacuo to give methyl 4-benzyloxycarbonyl-7-hy-
droxy-4-azaheptanoate (20) as colorless oil (11.3 g, 95.8%).
1H NMR (CDCl3) ꢁ 1.71 (2H, m, C6H2), 2.59 (2H, m, C2H2),
3.38 (2H, m, C5H2), 3.46 (2H, m, C3H2), 3.55 (2H, m, C7H2),
3.65 (3H, s, CH3), 5.16 (2H, s, CH2/Z), 7.30–7.40 (5H, m, Ph/
Z). MALDI TOF-MS: found m=z 296.2 [M + H]þ (calcd for
C15H21NO5 + H: 296.2).
4-azaheptanoic acid (23) in a quantitative yield.
To a solution of 23 obtained above in DMF (70 mL) were added
HONSu (1.43 g, 12.5 mmol) and EDC HCl (2.40 g, 12.5 mmol),
and the mixture was stirred overnight at r. t. The reaction mixture
was worked up in a similar manner as described in the preparation
of 18 to give 24 (4.53 g, 80.2%) as colorless oil. The thus-obtained
crude product was used for the next reaction without further puri-
fication.
ꢅ
4-Benzyloxycarbonylamino-1-butanol (25). To a solution of
4-amino-1-butanol (13) (5.00 g, 56.1 mmol) in acetone (25 mL)
were added TEA (6.81 g, 67.3 mmol) and ZONSu (14.4 g, 61.7
mmol). The mixture was stirred for 7 h at r. t., and the insoluble
material was filtered off. The filtrate was concentrated in vacuo,
and the residue was dissolved in AcOEt (100 mL). The solution
was washed with 1 M HCl (20 mL ꢃ 3), saturated aqueous
NaHCO3 (20 mL ꢃ 3), and brine (30 mL ꢃ 3). The organic layer
was dried over anhydrous MgSO4, and then concentrated in vacuo.
The thus-obtained crude crystalline product was recrystallized
from AcOEt and hexane to give 25 as colorless crystals (11.6 g,
To a solution of the thus-obtained 20 (11.0 g, 37.4 mmol) in tol-
uene (160 mL) were added TEA (4.15 g, 41.0 mmol) and MsCl
ꢄ
(4.28 g, 37.4 mmol) at 0 C, and the solution was stirred for 2 h
ꢄ
at 0 C. To the reaction mixture were added NaN3 (19.5 g, 299
ꢄ
1
mmol) and TBAB (3.58 g, 11.2 mmol) in H2O (160 mL) under
heating at 80 ꢄC. After stirring overnight at 80 ꢄC, the aqueous lay-
er was removed from the mixture, and the organic layer was wash-
ed with 10% citric acid (20 mL ꢃ 3), saturated aqueous NaHCO3
(20 mL ꢃ 3), and brine (20 mL ꢃ 3). The organic layer was dried
over anhydrous MgSO4, and the solvent was then removed in
vacuo. The thus-obtained crude product was purified by silica-
gel column chromatography (Art. 7734, 180 g, 2 ꢃ 70 cm, hexane:
AcOEt = 4:1). The fractions containing the desired product were
combined and concentrated in vacuo to give 21 as colorless oil
(7.94 g, 66.3%). 1H NMR (CDCl3) ꢁ 1.81 (2H, m, C6H2), 2.60
(2H, m, C2H2), 3.31 (2H, m, C5H2), 3.37 (2H, t, C3H2), 3.55
(2H, t, C7H2), 3.67 (3H, s, CH3), 5.13 (2H, s, CH2/Z), 7.26–7.40
(5H, m, Ph/Z). MALDI TOF-MS: found m=z 293.3 (M ꢁ N2 +
H)þ (calcd for C15H20N4O4 ꢁ N2 + H: 293.1).
92.8%); mp 78–79 C. H NMR (300 MHz, CDCl3) ꢁ 1.59 (4H,
m, C2H2, C3H2), 3.23 (2H, m, C4H2), 3.66 (2H, m, C1H2), 5.09
(2H, s, CH2/Z), 7.34 (5H, m, Ph/Z). MALDI TOF-MS: found
m=z 224.1 (M + H)þ (calcd for C12H17NO3 + H: 224.1), 246.1
(M + Na)þ (calcd for C12H16NO3 + Na: 246.1).
4-Azido-N-benzyloxycarbonylbutylamine (26). To a solution
of 25 (4.00 g, 17.9 mmol) in toluene (100 mL) were added TEA
ꢄ
(1.99 g, 19.7 mmol) and MsCl (2.05 g, 17.9 mmol) at 0 C, and
the mixture was stirred for 2 h at 0 ꢄC. To the solution were added
NaN3 (4.65 g, 71.6 mmol) and TBAB (0.577 g, 1.79 mmol) in H2O
(45 mL) under heating at 70 ꢄC. After the mixture was additionally
stirred for 24 h at 70 ꢄC, the organic layer separated from aqueous
layer was washed with 10% citric acid (15 mL ꢃ 3), saturated
aqueous NaHCO3 (15 mL ꢃ 3), and brine (15 mL ꢃ 3). The organ-
ic layer was dried over anhydrous MgSO4, and then concentrated
in vacuo. The thus-obtained crude product was purified by silica-
gel column chromatography (Art. 7734, 220 g, 4 ꢃ 100 cm, hex-
ane:AcOEt = 4:1). The fractions containing the desired product
were combined, and concentrated in vacuo to give 26 as colorless
oil (4.24 g, 95.6%). 1H NMR (CDCl3) ꢁ 1.60 (4H, m, C2H2, C3H2),
3.22 (2H, m, C4H2), 3.30 (2H, m, C1H2), 5.10 (2H, s, CH2/Z), 7.34
(5H, m, Ph/Z). MALDI TOF-MS: found m=z 271.3 (M + Na)þ
(calcd for C12H16N4O2 + Na: 271.1).
Methyl 4,N7-Bis(benzyloxycarbonyl)-7-amino-4-azahepta-
noate (22). To a solution of 21 (5.28 g, 16.5 mmol) in THF (40
mL) and H2O (4 mL) was added PPh3 (4.76 g, 18.1 mmol) at 0
ꢄC. The reaction mixture was stirred for 1 h at 0 ꢄC and addition-
ally for 30 h at r. t., and concentrated in vacuo. The residue was
dissolved in diethyl ether (150 mL), and extracted with 1 M HCl
(10 mL ꢃ 3). The acidic aqueous extract was lyophilized, and
the residue was dissolved in THF (20 mL). To a solution basified
with TEA (1.87 g, 18.4 mmol) was added dropwize ZCl (3.09 g,
18.1 mmol) over a 30 minute-period. The reaction mixture was
stirred overnight at r. t., and worked up in a similar manner as de-
scribed in the preparation of 16. The crude product was purified by
silica-gel column chromatography (Art. 7734, 38 g, 1 ꢃ 40 cm,
hexane:AcOEt = 5:1). The fractions containing the desired product
were combined, and concentrated in vacuo to give 22 as colorless
oil (5.08 g, 71.8%). 1H NMR (CDCl3) ꢁ 1.70 (2H, m, C6H2), 2.56
(2H, m, C2H2), 3.15 (2H, m, C5H2), 3.35 (2H, m, C3H2), 3.51 (2H,
t, C7H2), 3.65 (3H, s, CH3), 5.09 (2H, s, CH2/Z), 5.12 (2H, s, CH2/
Z), 7.25–7.40 (10H, m, Ph/Z). MALDI TOF-MS: found m=z 443.5
(M + H)þ (calcd for C24H30N2O6 + H: 443.2), 465.4 (M + Na)þ
(calcd for C24H30N2O6 + Na: 465.2).
N-Benzyloxycarbonyl-1,4-butanediamine
(27). To a solution of 26 (4.00 g, 29.7 mmol) in THF (30 mL)
Hydrochloride
and H2O (2 mL) was added PPh3 (5.06 g, 19.3 mmol) at 0 ꢄC.
ꢄ
The reaction mixture was stirred for 2 h at 0 C and additionally
for 20 h at r. t., and then concentrated in vacuo. The residue was dis-
solved in AcOEt (100 mL), and the solution was extracted with 10%
citric acid (30 mL ꢃ 3). The extract was washed with diethyl ether
(20 mL ꢃ 3), and then basified with 2 M NaOH; the basic aqueous
solution was extracted with CHCl3 (20 mL ꢃ 6). The extract was
dried over anhydrous MgSO4, and then concentrated in vacuo. The
residue was dissolved in 1 M HCl, and the solution was concentrat-
ed in vacuo to give 25 as colorless crystals (3.57 g, quant.) that
were used for the next reaction without further purification; mp
4,N7-Bis(benzyloxycarbonyl)-7-amino-4-azaheptanoic Acid
Succinimidyl Ester [Z-Apa(Z)-ONSu] (24). Methyl 4,N7-bis-
(benzyloxycarbonyl)-7-amino-4-azaoctanoate (22) (4.70 g, 11.0
mmol) was dissolved in 1 M NaOH (15 mL) ꢄand MeOH (15
mL), and the mixture was stirred for 1.5 h at 60 C. The reaction
mixture was worked up in a similar manner as described in the
preparation of 17 to give of 4,7-bis(benzyloxycarbonyl)-7-amino-
188–190 C (decomp.). H NMR (CD3OD) ꢁ 1.41 (4H, m, C2H2,
C3H2), 2.58 (2H, m, C4H2), 3.01 (2H, m, C1H2), 4.78 (2H, s,
CH2/Z), 7.20 (each 1H, brs, CH/Z). MALDI TOF-MS: found
m=z 223.0 (M + H)þ (calcd for C12H18N2O2 + H: 223.1).
3,N7-Bis(benzyloxycarbonyl)-7-amino-3-azaheptanoic Acid
[Z-Abg(Z)-OH] (28). N-Benzyloxycarbonyl-1,4-butanediamine
hydrochloride (27) (1.00 g, 3.86 mmol) in CH2Cl2 (30 mL) and
ꢄ
1

336 Bull. Chem. Soc. Jpn., 77, No. 2 (2004)
Structure Activity Relationships of NPTX-594
ꢀ
MeOH (10 mL) was added to glyoxylic acid monohydrate (0.237 g,
2.57 mmol), and the mixture was stirred for 15 min at r. t. To the
solution was added dropwise NaB(OAc)3H (1.69 g, 7.72 mmol) in
CH2Cl2 (20 mL) at r. t. over a 30-minute period, and the mixture
was additionally stirred for 5.5 h. The reaction mixture was con-
centrated in vacuo, and the residue was dissolved in saturated aque-
ous NaHCO3 (60 mL); the solution was washed with CH2Cl2 (20
mL ꢃ 3). To the basic aqueous solution was added ZONSu (0.599
g, 2.57 mmol) in MeOH (20 mL), and the mixture was stirred for
23 h at r. t. The reaction mixture was acidified with 1 M HCl, and
the acidic solution was extracted with AcOEt (30 mL ꢃ 3). The or-
ganic layer was dried over anhydrous MgSO4, and then concentrat-
ed in vacuo. The crude product was purified by silica-gel column
chromatography (Art. 7734, 100 g, 2 ꢃ 70 cm, CHCl3:MeOH =
9:1). The fractions containing the desired product were combined,
and concentrated in vacuo to give 28 as colorless oil (0.422 g,
39.3%). 1H NMR (CDCl3) ꢁ 1.52 (4H, m, C5H2, C6H2), 3.15
(2H, m, C7H2), 3.35 (2H, m, C4H2), 3.96 and 4.00 (each 1H, brs,
C1H2), 5.07 (2H, s, CH2/Z), 5.11 and 5.14 (each 1H, brs, CH2/
Z), 7.21–7.38 (10H, m, Ph/Z). MALDI TOF-MS: found m=z
437.6 (M + Na)þ (calcd for C22H26N2O6 + Na: 437.2).
4,8-Bis(benzyloxycarbonyl)-N12-{N -[2,4-bis(benzyloxy)-
phenylacetyl]asparaginyl}-N1-(2,2,2-trichloroethoxycarbonyl)-
4,8-diaza-1,12-dodecanediamine (32). To a solution of 31 (1.00
g, 2.91 mmol) in CH2Cl2 (10 mL) was added TFA (10 mL), and the
solution was stirred for 1.5 h at r. t. The reaction mixture was con-
centrated in vacuo, and the residue was dissolved in DMF (50 mL);
the solution was neutralized with TEA. To the solution were added
TEA (0.586 g, 5.82 mmol) and 2,4-bis(benzyloxy)phenylacetic
acid succinimidyl ester [Dhpa(Bzl)2-ONSu]20,23,24 (1.24 g, 2.91
mmol). The reaction mixture was stirred for 22 h at r. t., and then
concentrated in vacuo. The residue was dissolved in CHCl3 (250
mL), and the solution was washed with 10% citric acid (50 mL
ꢃ 3) and saturated aqueous NaHCO3 (50 mL ꢃ 2). The organic
layer was dried over anhydrous MgSO4, and then concentrated
in vacuo. The thus-obtained crude product was purified by silica-
gel column chromatography (Art. 9385, 208 g, 3:3 ꢃ 50 cm,
CHCl3:MeOH = 97:3). The fractions containing the desired prod-
uct were combined, and concentrated in vacuo to give 32 as color-
less crystals (3.10 g, 97.8%); mp 139–140 ꢄC. 1H NMR (CDCl3) ꢁ
1.28 (2H, m, C10H2/Dada), 1.38 (2H, m, C11H2/Dada), 1.56–1.80
(4H, m, {C2H2, C6H2}/Dada), 2.24 and 2.73 (each 1H, dd, ꢀ-
CH2/Asn, J ¼ 15:6, 6.6 Hz), 3.17 (12H, m, {C1H2, C3H2, C5H2,
C7H2, C9H2, C12H2}/Dada), 3.58 (2H, s, CH2/Dhpa), 4.61 (1H,
m, ꢂ-CH/Asn), 4.71 (2H, s, CH2/Troc), 5.01 (2H, s, CH2/Z),
5.07 and 5.09 (each 1H, s, CH2/Z), 6.61 (1H, dd, C3H/Dhpa,
J ¼ 8:4, 2.4 Hz), 6.64 (1H, d, C5H/Dhpa, J ¼ 2:4 Hz), 7.13
(1H, d, C6H/Dhpa, J ¼ 8:4 Hz), 7.20–7.42 (20H, m, Ph/Z and
Ph/Bzl). HRFAB-MS: found m=z 1089.3655 (M + H)þ (calcd
for C55H63Cl3N6O11 + H: 1089.3699).
3,N7-Bis(benzyloxycarbonyl)-7-amino-3-azaheptanoic Acid
Succinimidyl Ester [Z-Abg(Z)-ONSu] (29). To a solution of
28 (0.533 g, 1.29 mmol) in DMF (15 mL) were added HONSu
(0.140 g, 1.42 mmol) and EDC HCl (0.273 g, 1.42 mmol), and
ꢅ
the mixture was stirred for 11 h at r. t. The reaction mixture was
worked up in a similar manner as described in the preparation of
18 to give 29 as colorless crystals (0.530 g, 80.0%). The thus-ob-
tained crude product was used for the next reaction without further
purification. H NMR (CDCl3) ꢁ 1.55 (4H, m, C5H2, C6H2), 2.79
N12-[N -(2,4-Dihydroxyphenylacetyl)asparaginyl]-4,8-di-
1
ꢀ
(4H, m, CH2/ONSu), 3.16 and 3.20 (each 1H, m, C7H2), 3.37
(2H, m, C4H2), 4.30 and 4.37 (each 1H, brs, C1H2), 5.08 (2H, s,
CH2/Z), 5.15 and 5.17 (each 1H, brs, CH2/Z), 7.30–7.36 (10H,
m, Ph/Z). MALDI TOF-MS: found m=z 534.4 (M + Na)þ (calcd
for C26H29N3O8 + Na: 534.5).
aza-1,12-dodecanediamine [Des-Lys-NPTX-594] Tris-trifluo-
roacetate (2). To a solution of 32 (0.700 g, 0.640 mmol) in
90% AcOH (50 mL) was added Zn dust (1.28 g, 19.1 mmol),
and the suspension was stirred for 5.5 h at r. t. The insoluble ma-
terials were filtered off, and the filtrate was concentrated in vacuo;
the residue was dissolved in MeOH (15 mL) and AcOH (30 mL).
To the solution was added Pd-black (200 mg), and hydrogen was
gently introduced into the suspension under stirring for 7 h at r.
t. After the catalyst was filtered off, the filtrate was concentrated
in vacuo. The residue was finally purified by preparative RPHPLC
(10–40% CH3CN containing 0.1% TFA–H2O containing 0.1%
TFA; flow rate: 8.0 mL minꢁ1). The fractions containing the de-
sired compound were combined, and lyophilized to give 2 as a
powdery 3TFA salt (128 mg, 43.2%). 1H NMR (D2O) ꢁ 1.14–
1.37 (4H, m, {C10H2, C11H2}/Dada), 1.94 (4H, m, {C2H2,
C6H2}/Dada), 2.58 and 2.65 (each 1H, dd, ꢀ-CH2/Asn,
J ¼ 15:5, 7.5 Hz), 2.75–3.21 (12H, m, {C1H2, C3H2, C5H2, C7H2,
C9H2, C12H2}/Dada), 3.36 and 3.44 (each 1H, d, CH2/Dhpa, J ¼
15:6 Hz), 3.86 (1H, dd, ꢂ-CH/Asn, J ¼ 7:5, 7.5 Hz), 6.31 (2H, m,
Ph-{C3H, C5H}/Dhpa, J ¼ 8:7 Hz), 6.95 (1H, d, Ph-C6H/Dhpa,
J ¼ 8:7 Hz). HRFAB-MS: found m=z 467.2970 (M + H)þ (calcd
for C22H38N6O5 + H: 467.2982).
ꢀ
4,8-Bis(benzyloxycarbonyl)-N12-(N -t-butoxycarbonylas-
paraginyl)-N1-(2,2,2-trichloroethoxycarbonyl)-4,8-diaza-1,12-
dodecanediamine (31). To a solution of 4,8-bis(benzyloxycar-
bonyl)-N1-(2,2,2-trichloroethoxycarbonyl)-N12-(t-butoxycarbon-
yl)-4,8-diaza-1,12-dodecanediamine (30)17 (2.66 g, 3.57 mmol) in
CH2Cl2 (10 mL) was added TFA (10 mL), and the solution was
stirred for 30 min at r. t. The reaction mixture was concentrated
in vacuo, and the residue was dissolved in DMF (30 mL); the solu-
tion was neutralized with TEA. To the solution were added TEA
(0.719 g, 7.14 mmol) and Boc-Asn-ONp (1.52 g, 4.30 mmol),
and the mixture was stirred for 1 h at r. t. The reaction mixture
was concentrated in vacuo, and the residue was dissolved in AcOEt
(50 mL). The solution was washed with 10% citric acid (10 mL ꢃ
3), saturated aqueous NaHCO3 (10 mL ꢃ 6), and brine (10 mL ꢃ
3). The organic layer was dried over anhydrous MgSO4, and then
concentrated in vacuo. The crude product was purified by silica-gel
column chromatography (Art. 7734, 80 g, 2 ꢃ 47 cm, AcOEt and
then CHCl3:MeOH = 24:1). The fractions containing the desired
product were combined, and concentrated in vacuo to give 31 as
colorless crystals (2.77 g, 90.3%); mp 83–84 ꢄC. 1H NMR (CDCl3)
ꢁ 1.45 (9H, s, (CH3)3C/Boc), 1.45–1.74 (8H, m, {C2H2, C6H2,
C10H2, C11H2}/Dada), 2.52 and 2.91 (each 1H, dd, ꢀ-CH2/Asn,
J ¼ 15:6, 6.6 Hz), 3.20 (12H, m, {C1H2, C3H2, C5H2, C7H2, C9H2,
C12H2}/Dada), 4.43 (1H, m, ꢂ-CH/Asn), 4.71 (2H, s, CH2/Troc),
5.11 (4H, s, CH2/Z), 7.27–7.37 (10H, m, Ph/Z). HRFAB-MS:
found m=z 859.2934 (M + H)þ (calcd for C38H53Cl3N6O10 + H:
859.2967).
ꢀ
4,8-Bis(benzyloxycarbonyl)-N12-{N -[2,4-bis(benzyloxy)-
phenylacetyl]asparaginyl}-4,8-diaza-1,12-dodecanediamine
Acetate (33). To a solution of 32 (1.50 g, 1.38 mmol) in 90%
AcOH (50 mL) was added Zn dust (2.69 g, 41.1 mmol), and the
suspension was stirred for 24 h at r. t. H2S gas generated from solid
H2S (12.6 g, 14.1 mmol) was introduced to the suspension under
stirring for 1.5 h at r. t. After releasing excess H2S by introducing
CO2 generated from dry ice, the insoluble materials were filtered
off through celite 545@, and the filtrate was concentrated in vacuo.
The residue was dissolved in 1,4-dioxane (100 mL), and the solu-

T. Wakamiya et al.
Bull. Chem. Soc. Jpn., 77, No. 2 (2004)
337
tion was lyophilized to give 33 as a powdery monoacetate (1.28 g,
96.2%). The thus-obtained crude product was used for the next
reaction without further purification.
150 g, 3 ꢃ 50 cm, CHCl3:MeOH = 97:3) to give 42 as colorless
crystals (0.895 g, 74.6%); mp 125–128 ꢄC. 1H NMR (CDCl3) ꢁ
1.22–1.82 (12H, m, {C2H2, C6H2, C10H2, C11H2}/Dada, {C2H2,
C6H2}/Apa), 2.25 and 2.71 (each 1H, dd, ꢀ-CH2/Asn, J ¼ 15:5,
6.0 Hz), 2.89–3.41 (18H, m, {C1H2, C3H2, C5H2, C7H2, C9H2,
C12H2}/Dada, {C3H2, C5H2, C7H2}/Apa), 3.57 (2H, s, CH2/
Dhpa), 4.60 (1H, m, ꢂ-CH/Asn), 5.01 (2H, s, CH2/Z), 5.07
(10H, m, CH2/Z and CH2/Bzl), 6.56 (1H, dd, Ph-C3H/Dhpa,
J ¼ 8:4, 2.1 Hz), 6.64 (1H, d, Ph-C5H/Dhpa, J ¼ 2:1 Hz), 7.12
(1H, d, Ph-C6H/Dhpa, J ¼ 8:4 Hz), 7.26–7.43 (30H, m, Ph/Z
and Ph/Bzl). HRFAB-MS: found m=z 1311.6293 (M + H)þ (calcd
for C74H86N8O14 + H: 1311.6342).
4,8-Bis(benzyloxycarbonyl)-N1-[4,N8-bis(benzyloxycarbon-
ꢀ
yl)-8-amino-4-azaoctanoyl]-N12-{N -[2,4-bis(benzyloxy)phen-
ylacetyl]asparaginyl}-4,8-diaza-1,12-dodecanediamine
(41).
(General procedure 1: Coupling of the terminal amino acid resi-
due by the active ester method). To a solution of 33 (0.300 g, 0.308
mmol) in DMF (15 mL) were added TEA (0.0854 g, 0.616 mmol)
and Z-Pua(Z)-ONSu (18) (0.203 g, 0.370 mmol), and the mixture
was stirred for 18 h at r. t. The reaction mixture was concentrated in
vacuo, and the residue was dissolved in CHCl3 (300 mL). The so-
lution was washed with 10% citric acid (30 mL ꢃ 3) and saturated
aqueous NaHCO3 (50 mL ꢃ 3). The organic layer was dried over
anhydrous MgSO4, and then the solvent was removed in vacuo.
The crude product was purified by silica-gel column chromatogra-
phy (Art. 9385, 23.5 g, 1 ꢃ 45 cm, CHCl3 to CHCl3:MeOH =
91:9). The fractions containing the desired product were combined,
and concentrated in vacuo to give 41 as colorless crystals (0.368 g,
90.1%); mp 112–115 ꢄC. 1H NMR (CDCl3) ꢁ 1.26 (4H, m, {C10H2,
C11H2}/Dada), 1.43 (2H, m, C6H2/Pua), 1.53 (2H, m, C7H2/Pua),
1.73 (4H, m, {C2H2, C6H2}/Dada), 2.45 (2H, m, C2H2/Pua), 2.25
and 2.71 (each 1H, dd, ꢀ-CH2/Asn, J ¼ 15:6, 6.6 Hz), 2.85–3.38
(16H, m, {C1H2, C3H2, C5H2, C7H2, C9H2, C12H2}/Dada, {C3H2,
C5H2}/Pua), 3.51 (2H, m, C8H2/Pua), 3.57 (2H, s, CH2/Dhpa),
4.59 (1H, m, ꢂ-CH/Asn), 5.01 (2H, s, CH2/Z), 5.08 (10H, m,
CH2/Z and CH2/Bzl), 6.56 (1H, dd, Ph-C3H/Dhpa, J ¼ 8:1, 2.1
Hz), 6.64 (1H, d, Ph-C5H/Dhpa, J ¼ 2:1 Hz), 7.12 (1H, d, Ph-
C6H/Dhpa, J ¼ 8:1 Hz), 7.26–7.43 (30H, m, Ph/Z and Ph/Bzl).
HRFAB-MS: found m=z 1325.6434 (M + H)þ (calcd for
C75H88N8O14 + H: 1325.6498).
ꢀ
N1-(7-Amino-4-azaheptanoyl)-N12-[N -(2,4-dihydroxyphen-
ylacetyl)asparaginyl]-4,8-diaza-1,12-dodecanediamine {Apa-
[des-Lys-(NPTX-594)]} Tetrakis-trifluoroacetate (4).
Com-
pound 42 (200 mg, 0.152 mmol) in MeOH (15 mL) and AcOH
(30 mL) was hydrogenated in the presence of Pd-black (0.200 g)
as a catalyst, and worked up in a similar manner as described in
General procedure 2 to give 4 as white powdery substance (67.7
mg, 62.7%). 1H NMR (D2O) ꢁ 1.35 (4H, m, {C10H2, C11H2}/Da-
da), 1.80 (2H, m, C2H2), 1.95 (4H, m, C6H2/Dada, C6H2/Apa),
2.62 (2H, m, C2H2/Apa), 2.64 (2H, d, ꢀ-CH2/Asn, J ¼ 6:6 Hz),
2.80–3.14 (14H, m, {C3H2, C5H2, C7H2, C9H2}/Dada, {C3H2,
C5H2, C7H2}/Apa), 3.12–3.23 (4H, m, {C1H2, C12H2}/Dada),
3.39 and 3.47 (each 1H, d, CH2/Dhpa, J ¼ 15:3 Hz), 4.48 (1H,
t, ꢂ-CH/Asn, J ¼ 6:6 Hz), 6.35 (2H, m, Ph-{C3H, C5H}/Dhpa),
6.99 (1H, m, Ph-C6H/Dhpa). HRFAB-MS: found m=z 595.3928
(M + H)þ (calcd for C28H51N8O6 + H: 595.3932).
4,8-Bis(benzyloxycarbonyl)-N1-[3,N7-bis(benzyloxycarbon-
ꢀ
yl)-7-amino-3-azaheptanoyl]-N12-{N -[2,4-bis(benzyloxy)phen-
ylacetyl]asparaginyl}-4,8-diaza-1,12-dodecanediamine
(43).
ꢀ
N1-(8-Amino-4-azaoctanoyl)-N12-[N -(2,4-dihydroxyphen-
To a solution of 33 (0.120 g, 0.117 mol) in DMF (30 mL) were add-
ed TEA (0.185 g, 1.83 mmol) and Z-Abg(Z)-ONSu (29) (0.0663 g,
0.129 mmol), and the mixture was worked up in a similar manner
as described in General procedure 1. The crude product was puri-
fied by silica-gel column chromatography (Art. 9385, 14 g, 1 ꢃ 25
cm, CHCl3:MeOH = 20:1) to give 43 as colorless crystals (0.106 g,
69.9%); mp 119–122 ꢄC. 1H NMR (CDCl3) ꢁ 1.26–1.82 (12H, m,
{C2H2, C6H2, C10H2, C11H2}/Dada, {C5H2, C6H2}/Abg), 2.25
and 2.71 (each 1H, dd, ꢀ-CH2/Asn, J ¼ 15:9, 7.2 Hz), 2.83–
3.41 (18H, m, {C1H2, C3H2, C5H2, C7H2, C9H2, C12H2}/Dada,
{C2H2, C4H2, C7H2}/Abg), 3.57 (2H, s, CH2/Dhpa), 3.87 (1H,
m, ꢂ-CH/Lys), 4.60 (1H, m, ꢂ-CH/Asn), 5.01 (2H, s, CH2/Z),
5.07 (10H, m, CH2/Z and CH2/Bzl), 6.56 (1H, dd, Ph-C3H/Dhpa,
J ¼ 8:4, 2.1 Hz), 6.64 (1H, d, Ph-C5H/Dhpa, J ¼ 2:1 Hz), 7.13
(1H, d, Ph-C6H/Dhpa, J ¼ 8:4 Hz), 7.31–7.42 (30H, m, Ph/Z
and Ph/Bzl). HRFAB-MS: found m=z 1311.6307 (M + H)þ (calcd
for C74H86N8O14 + H: 1311.6342).
ylacetyl)asparaginyl]-4,8-diaza-1,12-dodecanediamine {Pua-
[des-Lys-(NPTX-594)]} Tetrakis-trifluoroacetate (3). (General
procedure 2: Deprotection and purification of final compounds).
To a solution of 41 (30 mg, 22.6 mmol) in MeOH (1 mL) and
AcOH (2 mL) was added 10% Pd(OH)2-C (35 mg) as a catalyst,
and the suspension was stirred under an atmosphere of hydrogen
for 24 h at r. t., and the catalyst was filtered off, and the filtrate
was concentrated in vacuo. The residue was finally purified by
preparative RPHPLC (10–40% CH3CN containing 0.1% TFA–
H2O containing 0.1% TFA; flow rate: 8.0 mL minꢁ1). The frac-
tions containing the desired compound were combined and lyophi-
lized to give 3 as a white powdery substance (4.00 mg, 16.6%).
1H NMR (D2O) ꢁ 1.35 (4H, m, {C10H2, C11H2}/Dada), 1.58
(4H, m, {C6H2, C7H2}/Pua), 1.71 (2H, m, C6H2/Dada), 1.86
(2H, m, C2H2/Dada), 2.53 (2H, m, C2H2/Pua), 2.51 and 2.61
(2H, dd, ꢀ-CH2/Asn, J ¼ 15:6, 6.0 Hz), 2.70–3.02 (14H, m,
{C3H2, C5H2, C7H2, C9H2}/Dada, {C3H2, C5H2, C8H2}/Pua),
3.08–3.15 (4H, m, {C1H2, C12H2}/Dada), 3.31 and 3.40 (each
1H, d, CH2/Dhpa, J ¼ 15:9 Hz), 4.40 (1H, dd, ꢂ-CH/Asn,
J ¼ 6:0, 6.0 Hz), 6.27 (2H, m, Ph-{C3H, C5H}/Dhpa), 6.91 (1H,
m, Ph-C6H/Dhpa). HRFAB-MS: found m=z 609.4061 (M + H)þ
(calcd for C29H53N8O6 + H: 609.4088).
ꢀ
N1-(7-Amino-3-azaheptanoyl)-N12-[N -(2,4-dihydroxyphen-
ylacetyl)asparaginyl]-4,8-diaza-1,12-dodecanediamine {Abg-
[des-Lys-(NPTX-594)]} Tetrakis-trifluoroacetate (5).
Com-
pound 43 (21.1 mg, 16.1 mmol) in MeOH (1 mL) and AcOH (2
mL) was hydrogenated in the presence of 10% Pd(OH)2-C (20
mg) as a catalyst, and worked up in a similar manner as described
in General procedure 2 to give 5 as white powdery 4TFA salt (6.30
mg, 37.3%). 1H NMR (D2O) ꢁ 1.35 (4H, m, C5H2/Abg, C6H2/
Abg), 1.60 (4H, m, {C10H2, C11H2}/Dada), 1.73 (2H, m, C6H2/
Dada), 1.86 (2H, m, C2H2/Dada), 2.52 and 2.61 (each 1H, dd,
ꢀ-CH2/Asn, J ¼ 15:9, 7.2 Hz), 2.72–3.04 (14H, m, {C3H2, C5H2,
C7H2, C9H2}/Dada, {C2H2, C4H2, C7H2}/Abg), 3.15 (4H, m,
{C1H2, C12H2}/Dada), 3.31 and 3.39 (each 1H, d, CH2/Dhpa, J ¼
15:3 Hz), 3.70 (1H, m, ꢂ-CH/Lys), 4.40 (1H, dd, ꢂ-CH/Asn,
4,8-Bis(benzyloxycarbonyl)-N1-[4,N7-bis(benzyloxycarbon-
ꢀ
yl)-7-amino-4-azaheptanoyl]-N12-{N -[2,4-bis(benzyloxy)phen-
ylacetyl]asparaginyl}-4,8-diaza-1,12-dodecanediamine
(42).
To a solution of 33 (0.894 g, 0.917 mmol) in DMF (30 mL) were
added TEA (0.185 g, 1.83 mmol) and Z-Apa(Z)-ONSu (24)
(0.469g 0.917 mmol), and the mixture was worked up in a similar
manner as described in General procedure 1. The crude product
was purified by silica-gel column chromatography (Art. 9385,

338 Bull. Chem. Soc. Jpn., 77, No. 2 (2004)
Structure Activity Relationships of NPTX-594
ꢀ
J ¼ 7:2, 7.2 Hz), 6.56 (2H, m, Ph-{C3H, C5H}/Dhpa), 6.91 (1H,
m, Ph-C6H/Dhpa). HRFAB-MS: found m=z 595.3903 (M + H)þ
(calcd for C28H51N8O6 + H: 595.3932).
N1-6-Aminocaproyl-N12-[N -(2,4-dihydroxyphenylacetyl)-
asparaginyl]-4,8-diaza-1,12-dodecanediamine {Acp-[des-Lys-
(NPTX-594)]} Tris-trifluoroacetate (7). Compound 45 (60.0
mg, 56.0 mmol) in MeOH (5 mL) and AcOH (10 mL) was hydro-
genated in the presence of Pd-black (50 mg) as a catalyst, and
worked up in a similar manner as described in General procedure
2 to give 7 as a white powdery 3TFA salt (5.60 mg, 15.6%).
1H NMR (D2O) ꢁ 1.19 (2H, m, C4H2/Acp), 1.26–1.56 (8H, m,
{C10H2, C11H2}/Dada, {C3H2, C5H2}/Acp), 1.70 (2H, m,
C2H2/Dada), 1.86 (2H, m, C6H2/Dada), 2.09 (2H, t, C2H2/
Acp), 2.55 and 2.61 (2H, d, ꢀ-CH2/Asn, J ¼ 15:0, 7.5 Hz),
2.70–3.04 (10H, m, C6H2/Acp, {C3H2, C5H2, C7H2, C9H2}/Da-
da), 3.10 (2H, m, C12H2/Dada), 3.31 and 3.39 (each 1H, d,
CH2/Dhpa, J ¼ 15:6 Hz), 3.46 and 3.54 (each 1H, m, C1H2/Da-
da), 4.40 (1H, dd, ꢂ-CH/Asn, J ¼ 7:5, 7.5 Hz), 6.27 (2H, m, Ph-
{C3H, C5H}/Dhpa), 6.91 (1H, m, Ph-C6H/Dhpa). HRFAB-MS:
found m=z 580.3823 (M + H)þ (calcd for C28H49N7O6 + H:
580.3823).
ꢀ
4,8-Bis(benzyloxycarbonyl)-N1-[N ,Ng,Ng-tris(benzyloxy-
ꢀ
carbonyl)arginyl]-N12-{N -[2,4-bis(benzyloxy)phenylacetyl]-
asparaginyl}-4,8-diaza-1,12-dodecanediamine (44). To a solu-
tion of 33 (100 mg, 0.103 mmol) in DMF (5 mL) were added TEA
ꢂ
(23.0 mg, 0.227 mmol) and N ,Ng,Ng-tris(benzyloxycarbonyl)-
arginine [Z-Arg(Z)2-ONSu] (34) (76.1 mg, 0.370 mmol), and the
mixture was worked up in a similar manner as described in General
procedure 1. The crude product was purified by silica-gel column
chromatography (Art. 9385, 24 g, 1 ꢃ 45 cm, CHCl3 to
CHCl3:MeOH = 97:3ꢄ) to give 44 as colorless crystals (0.107 g,
1
70.4%); mp 132–136 C. H NMR (CDCl3) ꢁ 1.25–1.43 (4H, m,
{C10H2, C11H2}/Dada), 1.68 (8H, m, {C2H2, C6H2}/Dada, ꢀ-
CH2/Arg, ꢃ-CH2/Arg), 2.23 and 2.70 (each 1H, dd, ꢀ-CH2/
Asn, J ¼ 15:0, 6.0 Hz), 2.93–3.33 (14H, m, {C1H2, C3H2, C5H2,
C7H2, C9H2, C12H2}/Dada, ꢁ-CH2/Arg), 3.56 (2H, s, CH2/Dhpa),
3.97 (1H, m, ꢂ-CH/Arg), 4.59 (1H, m, ꢂ-CH/Asn), 5.02–5.21
(14H, m, CH2/Z ꢃ 5 and CH2/Bzl ꢃ 2), 6.55 (1H, dd, Ph-C3H/
Dhpa, J ¼ 8:1, 2.1 Hz), 6.63 (1H, d, Ph-C5H/Dhpa, J ¼ 2:1
Hz), 7.13 (1H, d, Ph-C6H/Dhpa, J ¼ 8:1 Hz), 7.20–7.42 (35H,
m, Ph/Z and Ph/Bzl). HRFAB-MS: found m=z 1473.6799 (M +
H)þ (calcd for C82H93N10O16 + H: 1473.6771).
4,8-Bis(benzyloxycarbonyl)-N1-(N -benzyloxycarbonylglyc-
yl)-N12-{N -[2,4-bis(benzyloxy)phenylacetyl]asparaginyl}-4,8-
ꢀ
ꢀ
diaza-1,12-dodecanediamine (46). To a solution of 33 (120 mg,
0.117 mmol) in DMF (15 mL) were added TEA (26.0 mg, 0.257
mmol) and N-benzyloxycarbonylglycine succinimidyl ester [Z-
Gly-ONSu] (36) (39.5 mg, 0.129 mmol), and the mixture was
worked up in a similar manner as described in General procedure
1. The crude product was purified by silica-gel column chromatog-
raphy (Art. 9385, 14 g, 1 ꢃ 25 cm, CHCl3 to CHCl3:MeOH =
20:1) give 46 as colorless crystals (89.5 mg, 69.4%), mp 112–
ꢀ
N1-Arginyl-N12-[N -(2,4-dihydroxyphenylacetyl)asparagin-
yl]-4,8-diaza-1,12-dodecanediamine
{Arg-[des-Lys-(NPTX-
594)]} Tetrakis-trifluoroacetate (6). Compound 44 (125 mg,
22.6 mmol) in MeOH (10 mL) and AcOH (20 mL) was hydrogen-
ated in the presence of Pd-black (50 mg) as a catalyst, and worked
up in a similar manner as described in General procedure 2 to give
6 as a white powdery 4TFA salt (14.8 mg, 14.7%). 1H NMR (D2O)
ꢁ 1.34 (4H, m, {C10H2, C11H2}/Dada), 1.46 (2H, m, ꢃ-CH2/Arg),
1.73 (4H, m, ꢀ-CH2/Arg, C2H2/Dada), 1.86 (2H, m, C6H2/Dada),
2.53 and 2.61 (2H, dd, ꢀ-CH2/Asn, J ¼ 14:4, 5.7 Hz), 2.70–2.93
(8H, m, {C3H2, C5H2, C7H2, C9H2}/Dada), 3.00–3.28 (6H, m,
{C1H2, C12H2}/Dada, ꢁ-CH2/Arg), 3.31 and 3.39 (each 1H, d,
CH2/Dhpa, J ¼ 15:6 Hz), 3.78 (1H, t, ꢂ-CH/Arg), 4.38 and
4.41 (1H, t, ꢂ-CH/Asn, J ¼ 5:7 Hz), 6.26 (2H, m, Ph-{C3H,
C5H}/Dhpa), 6.90 (1H, m, C6H/Dhpa). HRFAB-MS: found m=z
623.3961 (M + H)þ (calcd for C28H50N10O6 + H: 623. 3993).
4,8-Bis(benzyloxycarbonyl)-N1-[6-(benzyloxycarbonylami-
116 C. H NMR (CDCl3) ꢁ 1.18–1.43 (4H, m, {C10H2, C11H2}/
Dada), 1.49–1.75 (4H, m, {C2H2, C6H2}/Dada), 2.26 and 2.70
(each 1H, dd, ꢀ-CH2/Asn, J ¼ 15:3, 6.9 Hz), 2.85–3.33 (12H,
m, {C1H2, C3H2, C5H2, C7H2, C9H2, C12H2}/Dada), 3.56 (2H,
s, CH2/Dhpa), 3.82 (2H, s, CH2/Gly), 4.59 (1H, m, ꢂ-CH/Asn),
5.01 (2H, s, CH2/Z), 5.06 (8H, m, CH2/Z and CH2/Bzl), 6.55
(1H, dd, Ph-C3H/Dhpa, J ¼ 8:1, 2.4 Hz), 6.64 (1H, d, Ph-C5H/
Dhpa, J ¼ 2:4 Hz), 7.12 (1H, d, Ph-C6H/Dhpa, J ¼ 8:1 Hz),
7.22–7.43 (25H, m, Ph/Z and Ph/Bzl). HRFAB-MS: found m=z
1106.5216 (M + H)þ (calcd for C62H71N7O12 + H: 1106.5239).
ꢄ
1
N12-[N -(2,4-Dihydroxyphenylacetyl)asparaginyl]-N1-glyc-
yl-4,8-diaza-1,12-dodecanediamine
ꢀ
{Gly-[des-Lys-(NPTX-
594)]} Tris-trifluoroacetate (8). Compound 46 (192 mg, 0.173
mmol) in MeOH (10 mL) and AcOH (20 mL) was hydrogenated
in the presence of Pd-black (50 mg) as a catalyst, and worked up
in a similar manner as described in General procedure 2 to give
8 as a white powdery 3TFA salt (66.4 mg, 60.4%). 1H NMR
(D2O) ꢁ 1.39 (4H, m, {C10H2, C11H2}/Dada), 1.77 (2H, m,
C2H2/Dada), 1.91 (2H, m, C6H2/Dada), 2.60 and 2.68 (2H, dd,
ꢀ-CH2/Asn, J ¼ 14:7, 7.8 Hz), 2.74–3.18 (10H, m, {C3H2, C5H2,
C7H2, C9H2, C12H2}/Dada), 3.20 (2H, m, C1H2/Dada), 3.36 and
3.43 (each 1H, d, CH2/Dhpa, J ¼ 15:5 Hz), 3.66 (2H, s, ꢂ-CH2/
Gly), 4.40 (1H, dd, ꢂ-CH/Asn, J ¼ 7:8, 7.8 Hz), 6.31 (2H, m,
Ph-{C3H, C5H}/Dhpa), 6.95 (1H, m, Ph-C6H/Dhpa). HRFAB-
ꢀ
no)caproyl]-N12-{N -[2,4-bis(benzyloxy)phenylacetyl]aspara-
ginyl}-4,8-diaza-1,12-dodecanediamine (45). To a solution of
33 (120 mg, 0.117 mmol) in DMF (5 mL) were added TEA
(26.0 mg, 0.257 mmol) and 6-(benzyloxycarbonylamino)caproic
acid succinimidyl ester [Z-Acp-ONSu]18 (35) (46.6 mg, 0.129
mmol), and the mixture was worked up in a similar manner as de-
scribed in General procedure 1. The crude product was purified by
silica-gel column chromatography (Art. 9385, 14 g, 1 ꢃ 25 cm,
CHCl3 to CHCl3:MeOH = 20:1) to give 45 as colorless crystals
(0.106 g, 77.9%); mp 126–130 ꢄC. 1H NMR (CDCl3) ꢁ 1.20–
1.84 (14H, m, {C2H2, C6H2, C10H2, C11H2}/Dada, {C3H2, C4H2,
C5H2}/Acp), 2.15 (2H, m, C2H2/Acp), 2.23 and 2.71 (each 1H,
dd, ꢀ-CH2/Asn, J ¼ 15:3, 6.0 Hz), 2.87–3.33 (14H, m, {C1H2,
C3H2, C5H2, C7H2, C9H2, C12H2}/Dada, C6H2/Acp), 3.57 (2H,
s, CH2/Dhpa), 4.22 (1H, m, ꢂ-CH/Glu), 4.60 (1H, m, ꢂ-CH/
Asn), 5.01 (2H, s, CH2/Z), 5.08 (8H, m, CH2/Z and CH2/Bzl),
6.56 (1H, dd, Ph-C3H/Dhpa, J ¼ 8:1, 2.4 Hz), 6.64 (1H, d, Ph-
C5H/Dhpa, J ¼ 2:4 Hz), 7.12 (1H, d, Ph-C6H/Dhpa, J ¼ 8:1
Hz), 7.22–7.43 (25H, m, Ph/Z and Ph/Bzl). HRFAB-MS: found
m=z 1162.5859 (M + H)þ (calcd for C66H80N7O12 + H:
1162.5865).
MS: found m=z 524.3187 (M + H)þ (calcd for C24H42N7O6
+
H: 524.3197).
N1-(O-Benzyl-N -benzyloxycarbonylseryl)-4,8-bis(benzyl-
ꢀ
oxycarbonyl)-N12-{N -[2,4-bis (benzyloxy)phenylacetyl]aspar-
aginyl}-4,8-diaza-1,12-dodecanediamine (47). To a solution
of 33 (300 mg, 0.308 mmol) in DMF (10 mL) were added TEA
ꢀ
ꢂ
(68.6 mg, 0.678 mmol) and O-benzyl-N -benzyloxycarbonylserine
succinimidyl ester [Z-Ser(Bzl)-ONSu] (37) (145 mg, 0.339 mmol),
and the mixture was worked up in a similar manner as described in
General procedure 1. The crude product was purified by silica-gel

T. Wakamiya et al.
Bull. Chem. Soc. Jpn., 77, No. 2 (2004)
339
column chromatography (Art. 9385, 20 g, 1 ꢃ 40 cm,
CHCl3:MeOH = 97:3ꢄ) to give 47 as colorless crystals (313 mg,
MS: found m=z 614.3664 (M + H)þ (calcd for C31H47N7O6
+
H: 614.3666).
1
ꢁ
ꢀ
82.8%), mp 122–125 C. H NMR (DMSO-d6) ꢁ 1.14–1.42 (4H,
N1-(O -Benzyl-N -benzyloxycarbonylglutamyl)-4,8-bis(ben-
m, {C10H2, C11H2}/Dada), 1.48–1.70 (4H, m, {C2H2, C6H2}/Da-
da), 2.22–2.56 (2H, m, ꢀ-CH2/Asn), 2.80–3.20 (12H, m, {C1H2,
C3H2, C5H2, C7H2, C9H2, C12H2}/Dada), 3.40 (2H, s, CH2/
Dhpa), 3.56 (1H, m, ꢂ-CH/Ser), 4.21 (1H, m, ꢂ-CH/Asn), 4.34
(2H, m, ꢀ-CH2/Ser), 5.01–5.07 (12H, m, CH2/Phe, CH2/Z, and
CH2/Bzl), 6.51 (1H, d, Ph-C3H/Dhpa, J ¼ 8:1 Hz), 6.63 (1H, s,
Ph-C5H/Dhpa), 7.06 (1H, d, Ph-C6H/Dhpa, J ¼ 8:1 Hz), 7.20–
7.43 (30H, m, Ph/Z and Ph/Bzl). HRFAB-MS: found m=z
1226.5757 [M + H]þ (calcd for C70H79N7O13 + H: 1226.5814).
zyloxycarbonyl)-N12-{N -[2,4-bis(benzyloxy)phenylacetyl]as-
ꢀ
paraginyl}-4,8-diaza-1,12-dodecanediamine (49). To a solution
of 33 (300 mg, 0.308 mmol) in DMF (15 mL) were added TEA
ꢃ
ꢂ
(62.3 mg, 0.614 mmol) and O -benzyl-N -benzyloxycarbonylglu-
tamic acid succinimidyl ester [Z-Glu(OBzl)-ONSu] (39) (173 mg,
0.370 mmol), and the reaction mixture was worked up in a similar
manner as described in General procedure 1. The crude product
was purified by silica-gel column chromatography (Art. 9385, 23
g, 1 ꢃ 40 cm, CHCl3:MeOH = 98:2) to give 49 as colorless crys-
tals (255 mg, 65.2%); mp 129–132 ꢄC. 1H NMR (CDCl3) ꢁ 1.26–
1.38 (4H, m, {C10H2, C11H2}/Dada), 1.50–2.03 (6H, m, {C2H2,
C6H2}/Dada, ꢀ-CH2/Glu), 2.44 (2H, m, ꢃ-CH2/Glu), 2.24 and
2.71 (each 1H, dd, ꢀ-CH2/Asn, J ¼ 14:4, 6.3 Hz), 2.93–3.33
(12H, m, {C1H2, C3H2, C5H2, C7H2, C9H2, C12H2}/Dada), 3.57
(2H, s, CH2/Dhpa), 4.22 (1H, m, ꢂ-CH/Glu), 4.60 (1H, m, ꢂ-
CH/Asn,), 5.01 (2H, s, CH2/Z), 5.06 (10H, m, CH2/Z and CH2/
Bzl), 6.55 (1H, dd, Ph-C3H/Dhpa, J ¼ 8:6, 2.4 Hz), 6.63 (1H, d,
Ph-C5H/Dhpa, J ¼ 2:4 Hz), 7.12 (1H, d, Ph-C6H/Dhpa, J ¼ 8:6
Hz), 7.22–7.43 (30H, m, Ph/Z and Ph/Bzl). HRFAB-MS: found
m=z 1268.5916 (M + H)þ (calcd for C72H81N7O14 + H:
1268.5920).
ꢀ
N12-[N -(2,4-Dihydroxyphenylacetyl)asparaginyl]-N1-seryl-
4,8-diaza-1,12-dodecanediamine {Ser-[des-Lys-(NPTX-594)]}
Tris-trifluoroacetate (9). Compound 47 (50 mg, 0.408 mmol)
in MeOH (1 mL) and AcOH (2 mL) was hydrogenated in the pres-
ence of 10% Pd(OH)2-C (50 mg) as a catalyst, and worked up in a
similar manner as described in General procedure 2 to give 9 as a
white powdery 3TFA salt (4.20 mg, 18.6%). 1H NMR (D2O) ꢁ 1.35
(4H, m, {C10H2, C11H2}/Dada), 1.74 (2H, m, C2H2/Dada), 1.86
(2H, m, C6H2/Dada), 2.54 and 2.61 (each 1H, dd, ꢀ-CH2/Asn,
J ¼ 15:9, 6.3 Hz), 2.68–3.24 (12H, m, {C1H2, C3H2, C5H2, C7H2,
C9H2, C12H2}/Dada), 3.31 and 3.39 (each 1H, d, CH2/Dhpa, J ¼
15:6 Hz), 3.75 (2H, m, ꢀ-CH2/Ser), 3.86 (1H, m, ꢂ-CH/Ser), 4.38
and 4.41 (1H, t, ꢂ-CH/Asn, J ¼ 6:3 Hz), 6.27 (2H, m, Ph-{C3H,
C5H}/Dhpa), 6.91 (1H, m, Ph-C6H/Dhpa). HRFAB-MS: found
m=z 554.3278 [M + H]þ (calcd for C25H43N7O7 + H: 554.3302).
ꢀ
N12-[N -(2,4-Dihydroxyphenylacetyl)asparaginyl]-N1-glu-
tamyl-4,8-diaza-1,12-dodecanediamine {Glu-[des-Lys-(NPTX-
594)]} Tris-trifluoroacetate (11).
Compound 49 (245 mg,
ꢀ
4,8-Bis(benzyloxycarbonyl)-N1-(N -benzyloxycarbonyl-
0.193 mmol) in MeOH (10 mL) and AcOH (20 mL) was hydrogen-
ated in the presence of Pd-black (50 mg) as a catalyst, and worked
up in a similar manner as described in General procedure 2 to give
11 as a white powdery 3TFA salt (53.2 mg, 29.4%). 1H NMR
(D2O) ꢁ 1.41 (4H, m, {C10H2, C11H2}/Dada), 1.79 (2H, m,
C2H2/Dada), 1.91 (2H, m, C6H2/Dada), 2.04 (2H, dd, ꢀ-CH2/
Glu, J ¼ 6:9, 7.8 Hz), 2.37 (2H, t, ꢃ-CH2/Glu, J ¼ 7:8 Hz),
2.57 and 2.66 (2H, dd, ꢀ-CH2/Asn, J ¼ 15:0, 7.5 Hz), 2.75–3.20
(8H, m, {C3H2, C5H2, C7H2, C9H2}/Dada), 3.20–3.30 (4H, m,
{C1H2, C12H2}/Dada), 3.36 and 3.43 (each 1H, d, CH2/Dhpa, J ¼
15:6 Hz), 3.88 (1H, t, ꢂ-CH/Glu, J ¼ 6:9 Hz), 4.44 (1H, t, ꢂ-CH/
Asn, J ¼ 7:5 Hz), 6.31 (2H, m, Ph-{C3H, C5H}/Dhpa), 6.95 (1H,
m, Ph-C6H/Dhpa). HRFAB-MS: found m=z 596.3372 (M + H)þ
(calcd for C27H45N7O8 + H: 596.3408).
ꢀ
phenylalanyl)-N12-{N -[2,4-bis(benzyloxy)phenylacetyl]aspar-
aginyl}-4,8-diaza-1,12-dodecanediamine (48). To a solution
of 33 (300 mg, 0.308 mmol) in DMF (10 mL) were added TEA
ꢂ
(68.6 mg, 0.678 mmol) and N -benzyloxycarbonylphenylalanine
succinimidyl ester [Z-Phe-ONSu] (38) (122 mg, 0.339 mmol),
and it was worked up in a similar manner as described in General
procedure 1. The crude product was purified by silica-gel column
chromatography (Art. 9385, 20 g, 1 ꢃ 40 cm, CHCl3:MeOH =
97:3) to give 48 as colorless crystals (238 mg, 64.7%); mp 117–
120 ꢄC. 1H NMR (DMSO-d6) ꢁ 1.10–1.42 (4H, m, {C10H2,
C11H2}/Dada), 1.46–1.72 (4H, m, {C2H2, C6H2}/Dada), 2.22–
2.56 (2H, m, ꢀ-CH2/Asn), 2.88–3.20 (12H, m, {C1H2, C3H2,
C5H2, C7H2, C9H2, C12H2}/Dada), 3.40 (2H, s, CH2/Dhpa),
4.16 (1H, m, ꢂ-CH/Phe), 4.50 (1H, m, ꢂ-CH/Asn), 4.92–5.07
(12H, m, CH2/Phe, CH2/Z, and CH2/Bzl), 6.52 (1H, dd, Ph-
C3H/Dhpa, J ¼ 8:6, 2.4 Hz), 6.67 (1H, d, Ph-C5H/Dhpa, J ¼
2:4 Hz), 7.06 (1H, d, Ph-C6H/Dhpa, J ¼ 8:6 Hz), 7.20–7.43
(30H, m, Ph/Phe, Ph/Z, and Ph/Bzl). HRFAB-MS: found m=z
1196.5715 (M + H)þ (calcd for C69H78N7O12 + H: 1196.5708).
ꢂ
ꢀ
N1-(O -Benzyl-N -benzyloxycarbonylaspartyl-4,8-bis(ben-
zyloxycarbonyl)-N12-{N -[2,4-bis(benzyloxy)phenylacetyl]as-
ꢀ
paraginyl}-4,8-diaza-1,12-dodecanediamine (50). To a solution
of 33 (300 mg, 0.308 mmol) in DMF (10 mL) were added TEA
ꢃ
ꢂ
(68.6 mg, 0.678 mmol) and O -benzyl-N -benzyloxycarbonylas-
partic acid succinimidyl ester [Z-Asp(OBzl)-ONSu] (40) (154
mg, 0.339 mmol), and the reaction mixture was worked up in a
similar manner as described in General procedure 1. The crude
product was purified by silica-gel column chromatography (Art.
9385, 20 g, 1 ꢃ 40 cm CHCl3:MeOH = 97:3) to give 50 as color-
less crystal (0.338 mg, 87.6%); mp 121–125 ꢄC. 1H NMR (DMSO-
d6) ꢁ 1.15–1.43 (4H, m, {C10H2, C11H2}/Dada), 1.45–1.73 (4H, m,
{C2H2, C6H2}/Dada), 2.22–2.56 (4H, m, ꢀ-CH2/Asn, ꢀ-CH2/
Asp), 2.88–3.20 (12H, m, {C1H2, C3H2, C5H2, C7H2, C9H2,
C12H2}/Dada), 3.40 (2H, s, CH2/Dhpa), 4.36 (1H, m, ꢂ-CH/
Asp), 4.50 (1H, m, ꢂ-CH/Asn), 4.95–5.10 (12H, m, CH2/Z and
CH2/Bzl), 6.51 (1H, dd, Ph-C3H/Dhpa, J ¼ 8:6, 2.4 Hz), 6.66
(1H, d, Ph-C5H/Dhpa, J ¼ 2:4 Hz), 7.05 (1H, d, Ph-C6H/Dhpa,
J ¼ 8:6 Hz), 7.20–7.43 (30H, m, Ph/Z and Ph/Bzl). HRFAB-
ꢀ
N12-[N -(2,4-Dihydroxyphenylacetyl)asparaginyl]-N1-phen-
ylalanyl-4,8-diaza-1,12-dodecanediamine
{Phe-[des-Lys-
(NPTX-594)]} Tetrakis-trifluoroacetate (10). Compound 48
(50 mg, 0.418 mmol) in MeOH (1 mL) and AcOH (2 mL) was hy-
drogenated in the presence of 10% Pd(OH)2-C (50 mg) as a cata-
lyst, and worked up in a similar manner as described in General
procedure 2 to give 10 as a white powdery 3TFA salt (7.74 mg,
1
30.1%). H NMR (D2O) ꢁ 1.36 (4H, m, {C10H2, C11H2}/Dada),
1.53 (2H, m, C2H2/Dada), 1.85 (2H, m, C6H2/Dada), 2.58 (2H,
m, ꢀ-CH2/Asn), 2.70–3.18 (14H, m, {C1H2, C3H2, C5H2, C7H2,
C9H2, C12H2}/Dada, ꢀ-CH2/Phe), 3.30 and 3.39 (each 1H, d,
CH2/Dhpa, J ¼ 15:9 Hz), 3.97 (1H, t, ꢂ-CH/Phe), 4.40 (1H, t,
ꢂ-CH/Asn), 6.27 (2H, m, Ph-{C3H, C5H/}Dhpa), 6.90 (1H, m,
Ph-C6H/Dhpa), 7.10 (2H, m, Ph-{C3H, C5H}/Phe), 7.19–7.26
(3H, m, Ph-C2H/Phe, Ph-(C4H)/Phe, Ph-C6H/Phe). HRFAB-
MS: found m=z 1254.5704 (M + H)þ (calcd for C71H79N7O14
+
H: 1254.5763).

340 Bull. Chem. Soc. Jpn., 77, No. 2 (2004)
Structure Activity Relationships of NPTX-594
Am. Chem. Soc., 112, 6696 (1990).
ꢀ
N1-Aspartyl-N12-[N -(2,4-dihydroxyphenylacetyl)aspara-
ginyl]-4,8-diaza-1,12-dodecanediamine {Asp-[des-Lys-(NPTX-
594)]} Tris-trifluoroacetate (12). Compound 50 (30 mg, 23.9
mmol) in MeOH (1 mL) and AcOH (2 mL) was hydrogenated in
the presence of 10% Pd(OH)2-C (50 mg) as a catalyst, and worked
up in a similar manner as described in General procedure 2 to give
12 as a white powdery 3TFA salt (5.99 mg, 27.0%). 1H NMR
(D2O) ꢁ 1.36 (4H, m, {C10H2, C11H2})Dada), 1.72 (2H, m,
C2H2/Dada), 1.86 (2H, m, C6H2/Dada), 2.55 and 2.61 (2H, dd,
ꢀ-CH2/Asn, J ¼ 15:0, 7.5 Hz), 2.70 (2H, d, ꢀ-CH2/Asp, J ¼
6:0 Hz), 2.72–3.04 (10H, m, {C3H2, C5H2, C7H2, C9H2,
C12H2}/Dada), 3.09–3.28 (each 1H, m, C1H2/Dada), 3.31 and
3.39 (each 1H, d, CH2/Dhpa, J ¼ 15:6 Hz), 4.05 (1H, t, ꢂ-CH/
Asp, J ¼ 6:0 Hz), 4.40 (1H, d, ꢂ-CH/Asn, J ¼ 7:5 Hz), 6.27
(2H, m, Ph-{C3H, C5H}/Dhpa), 6.96 (1H, m, Ph-C6H/Dhpa).
13 G. B. Quistad, C. C. Reuter, W. S. Skinner, P. A. Dennis, S.
Suwanurupha, and E. W. Fu, Toxicon, 29, 329 (1991).
14 K. D. McCormich and J. Meinwald, J. Chem. Ecol., 19,
2411 (1993).
15 M. Hisada, T. Fujita, H. Naoki, Y. Itagaki, H. Irie, M.
Miyashita, and T. Nakajima, Toxicon, 36, 1115 (1998).
16 N. Kawai, A. Niwa, and T. Abe, Brain Res., 247, 169
(1982).
17 T. Wakamiya, A. Yamamoto, K. Kawaguchi, T. Kinoshita,
Y. Yamaguchi, Y. Itagaki, H. Naoki, and T. Nakajima, Bull. Chem.
Soc. Jpn., 74, 1743 (2001).
18 Abbreviations according to IUPAC-IUB commission, Eur.
J. Biochem., 138, 9 (1984), are used. Abg: N-(4-aminobutyl)-
glycine; Acp: 6-aminocaproic acid = 6-aminohexanoic acid;
AcOEt: ethyl acetate; Apa: N-(3-aminopropyl)-ꢀ-alanine; Arg: L-
arginine; Asn: L-asparagine; Boc: t-butoxycarbonyl; Bzl: benzyl;
DMF: N,N-dimethylformamide; DMSO: dimethyl sulfoxide;
HRFAB-MS: found m=z 582.3240 (M
C26H43N7O8 + H: 582.3251).
+
H)þ (calcd for
EDC HCl: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hy-
This work was supported in part by a grant from the Re-
search for the Future Program from the Japan Society for the
Promotion of Science (1998–2002), and a SUNBOR GRANT
from Suntory Institute for Bioorganic Research.
ꢅ
drochloride; Glu: L-glutamic acid; Gly: glycine; HRFAB-MS: high
resolution fast atom bombardment mass spectrometry; HONSu: N-
hydroxysuccinimide; Lys: L-lysine; MALDI TOF-MS: matrix as-
sisted laser desorption ionization time of flight mass spectrometry;
MsCl: methanesulfonyl chloride; Np: p-nitrophenyl (or 4-nitro-
phenyl); Phe: L-phenylalanine; Pua: N-(4-aminobutyl)-ꢀ-alanine;
RPHPLC: reversed-phase high-performance liquid chromatogra-
phy; Ser: L-serine; TBAB: tetrabutylammonium bromide; TEA:
triethylamine; TFA: trifluoroacetic acid; THF: tetrahydrofuran;
Troc: 2,2,2-trichloroethoxycarbonyl; TrocCl: 2,2,2-trichloro-
ethoxycarbonyl chloride; Z: benzyloxycarbonyl; ZCl: benzyloxy-
carbonyl chloride.
19 M. Miyashita, H. Sato, M. Matsushita, Y. Kusumegi, T.
Toki, A. Yoshikoshi, T. Yanami, Y. Kikuchi, C. Takasaki, T.
Nakajima, and H. Irie, Tetrahedron Lett., 33, 2837 (1992).
20 T. Chiba, T. Akizawa, M. Matsukawa, N. Nishi, N. Kawai,
and M. Yoshioka, Chem. Pharm. Bull., 44, 972 (1996).
21 P. G. Mattingly, Synthesis, 1990, 366.
References
#
A part of this work was presented at a) the 25th European
Peptide Symposium: T. Wakamiya, A. Yamamoto, K. Kawaguchi,
T. Kinoshita, Y. Yamaguchi, Y. Itagaki, H. Naoki, G. Corzo, and
T. Nakajima, ‘‘Peptides 2000,’’ ed by J. Martinez and J. Fehrentz,
EDK, Paris (2001), pp. 771–772, and b) the 37th Japanese Peptide
Symposium: T. Wakamiya, T. Kinoshita, Y. Yamaguchi, Y.
Itagaki, H. Naoki, G. Corzo, and T. Nakajima, ‘‘Peptides Science
2000,’’ ed by T. Shioiri, Japanese Peptide Society, Osaka (2001),
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1
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2
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3
4
5
24 M. Matsushita, T. Kanemura, S. Hatakeyama, H. Irie, T.
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6
7
8
26 Abg-[des-Lys-(NPTX-594)]28 shows stronger activity than
natural product.
9
T. Toki, T. Yasuhara, Y. Aramaki, Y. Hashimoto, K.
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27 In the case of NSTX-3 belonging to the type-D acylpoly-
amine toxin, i.e., Dhpa-Asn-NH(CH2)5NH Pua Arg, the re-
placement of the Arg residue with the neutral amino acid residues
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28 The analog is abbreviated as Xaa-[des-Lys-(NPTX-594)] in
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Lys residue in NPTX-594.
10 G. B. Quistad, S. Suwanurupha, M. A. Jarema, M. J.
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