Pergamon

0040-4039(95)00979-5

Tetrahedron Letters, Vol. 36, No. 29, pp. 5231-5234, 1995 Elsevier Science Ltd

Printed in Great Britain 0040-4039/95 $9.504-0.00

Tota l Synthesis of Nephi la tox in .7 (NPTX-7) , a New Neurotoxin of

J o r o Sp ide r (Nephila clavata)

Masayuki Matsushita, a Takanori Kanemura, a Susumi Hatakeyama, a Hiroshi Irie, a*

Takashi Toki, b and Masaaki Miyashita a*'f

aFaculty of Pharmaceutical Sciences, Nagasaki University, Nagasaki 852, Japan; bResearch Center,

Dalcel Chemical Industries, LTD., I-Iimeji 671-12, Japan

Abstract: The first and highly efficient total synthesis of Nephilatoxin-7 (NPTX-7), a new

neurotoxin of Joro spider (Nephila clavata), has been achieved by employing the azide strategy wherein

the three characteristic polyamine components of the toxin, cadaverine and bis-putreanine, were

efficiently constructed by the iterative use of key azide intermediates.

Spider toxins such as NSTX-3,1 JSTX-3,1 and Nephilatoxins (NPTXs) 2 have been

demonstrated to be potent and specific blocking agents of glutaminergic neuromuscular transmission and

are rapidly emerging as unique tools for understanding excitatory amino acid transmission and related

pharmacology. 2-4 The Nephilatoxins (NPTX-1-12) newly isolated from the Joro spider (Nephila clavata) 2 have also been shown to have potent activities on the mast cell degranulation and pesticide

activities. However very limited quantities of spider toxins have impeded their pharmacological

evaluation and ongoing biological studies. We have studied the chemical synthesis of spider toxins,

inter alia the NPTXs, and so far developed synthetic methodologies for NPTX-9 and 11, 5 NPTX-10

and 12, 6 and NPTX-87 in which key azide intermediates (2 and 3) were designed and employed for the

effective incorporation of the characteristic polyamine chains of these toxins. 8 We report herein the fast

and highly efficient total synthesis of NPTX-7 (1) based on the azide strategy in which key azide

intermediates were iteratively used in the construction of the characteristic polyamine moiety of the toxin.

NPTX-7 (1) has such unique structural features among ca. twenty Joro spider toxins isolated so far that

it is the only toxin containing an acidic amino acid, aspartic acid, as a component and also possesses the

novel polyamine chain comprising cadaverine (1,5-diaminopentane) and two molecules of putreanine (8-

amino-4-azaoctanoic acid). As shown in Fig. 1, NPTX-7 (1) consists of six components, i.e., indole-

3-acetic acid, omithine, aspartic acid, cadaverine, and two putreanine units. Crucial points in the

Fig. 1 HO

O H I01 " ~ C H H I01

~ N ~ N ~ N ~ N ,y.,"',,.,~ N ~ , 7 ' ~ N ~',,,,,,,,,,,,'~ N ~ N H 2 I ~ N ~ I O H i "~ H ~ ~ H H

2 NPTX-7 (1)

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5232

Scheme 1

B o c H N ~ N 3 Boc I

R O 2 c ~ N ~ N 3

3 R=CH 3 4 R = SuN

B o c H N ~ N 3

2

i, ii 93%

tBuO HO

FmocHN~/ N3 ii;~ ~v O

0 tBuQ O F m o c H N ~ N ~ N ~ N 3

H "

BocHN3 O

6

iii, v i t ,

97%

tBuQ H O C

~ N ~ N ~ N ~ N 3 ~ , , N ~ 0 L~ H ~

H BocHi- 7

tBuO. O

H o H ,Boo

vi, vii93% " ~ , , ~ N . ~ 5 --~ H O 5 a vi, vii67%

H BocH N "

8

tBuO 3= O

, O ( , , Boc O

~ . . ~ N ~ O ~ - O O Boc

H BocHN- 9

viii, ix ,, 1

Reagents and conditions: i, AcC1, MeOH; ii, Fmoc-Asp(OBut), EDC'HC1, HOBt, ipr2NEt, DMF; iii, Et2NH, CH2C12; iv, Fmoc-Orn(Boc), EDC.HC1, HOBt, DMF; v, Indole-3-acetic acid, EDC-HC1, HOBt, NMM, DMF; vi, H 2, Pd-BaSO 4, EtOH; vii, 4, DMF; viii, 10%Pd-C, HCO2NH 4, MeOH; ix, TFA, HSCH2CH2SH , CH2C12.

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synthesis of 1 are construction of the unique bis-putreanine moiety linked to cadaverine and introduction

of an acidic amino acid, aspartic acid. The successful synthetic route of I is shown in Scheme 1.

The synthesis started from 5-azido-l-N-Boc-aminopentane (2), a key azido compound readily

prepared from 5-amino- 1-pentanol. 5 After elimination of the Boc group of 2 with HC1 generated in situ

from acetyl chloride and methanol the resulting hydrochloride salts were condensed with N-(9-

fluorenylmethoxycarbonyl)-L-aspartic acid-~-t-butyl ester (Fmoc-Asp(OBut)) in the presence of 1-(3-

dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HC1), 1-hydroxybenzotriazole (HOBt),

and diisopropylethylamine in N,N-dimethylformamide (DMF) to give 5 in 93% yield. Subsequent

removal of the Fmoc group of 5 with diethylamine in CH2C12 followed by coupling with N~-(9-

fluorenylmethoxycarbonyl)-N~-(t-butyloxycarbonyl)-L-omithine (Fmoc-Om(Boc)) in DMF furnished

6 as colorless crystals (mp 133-134 °C) in 94% yield. Similar treatment of 6 with diethylamine in

CH2C12 and coupling of the resulting amine with indole-3-acetic acid in the presence of EDC.HC1,

HOBt, and N-methylmorpholine (NMM) in DMF yielded the left-half segment 7 (mp 135-136 °C)

having an indole-3-acetyl-omithinyl-asparatyl-cadaverine structure in excellent yield. Construction of

the bis-putreanine moiety of NPTX-7 (1), the crucial step in the present synthesis, was accomplished as

follows. Catalytic hydrogenation of the azide 7 over Pd-BaSO 4 in ethanol followed by condensation of

the resulting amine with succinimidyl 8-azido-N-Boc-4-azaoctanoate (4), the second key azido

compound readily derived from 3,5, 9 in DMF furnished 8 (nap 78-80 °C) in 93% yield. 10 The same

reaction sequence was repeated on the azide 8 to introduce the second putreanine molecule to afford 9 l0

which contains all the components of NPTX-7 (1). Finally, catalytic transfer hydrogenation of the

terminal azido group of 9 with 10% Pd-C and ammonium formate in MeOH followed by deprotection of

the three Boc groups and t-butyl ester with trifluoroacetic acid (TFA) in CH2C12 containing 1,2-

ethanedithiol cleanly produced 1 as TFA salts. The product ([~]D 18 -7.45 ° (c 0.51, H20)) was

identified with TFA salts of natural NPTX-7 by HPLC analyses (a TSKgel ODS-80TS column (4.6 x

150 mm), 12% CH3CN containing 0.1% TFA). In addition, all the spectral data of the synthetic

compound ( 500 MHz 1H-NMR and FAB-MS 773 (M+I)) were in agreement with the proposed

structure. 11 Thus the efficient total synthesis of NPTX-7 (1) was achieved by employing the azide

strategy. Biological evaluations of the synthetic compound and extensions of the methodology to other

spider toxins are in progress in our laboratory.

Acknowledgment: We are grateful to the Akiyama Foundation and the Tokyo Biochemical

Foundation for their financial supports. This work was also supported by a Grant-in-Aid for Scientific

Research on Priority Areas (No. 07229101), a Grant-in-Aid for Developmental Scientific Research (No.

04557100), a Grant-in-Aid for Co-operative Research (A) (No. 05303011), and a JSPS Research

Fellowship for Young Scientists (No. 2673) from the Ministry of Education, Science and Culture of

Japan.

tPresent address: Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060,

Japan.

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References and Notes

1 Y. Aramaki, T. Yasuhara, T. Higashijima, M. Yoshioka, A. Miwa, N. Kawai, and T. Nakajima,

Proc. Jpn. Acad.,1986, 62 0B), 359.

2 T. Told, T. Yasuhara, Y. Aramald, N. Kawai, and T. Nakajima, Biomedical Res., 1988, 9, 75; T.

Told, T. Yasuhara, Y. Aramaki, K. Osawa, A. Miwa, N. Kawai, and T. Nakajima, ibid., 1988, 9,

421.

3 N. Kawai, A. Miwa, M. Saito, H. Pan-Hou, and M. Yoshioka, J. Phsiol., Paris, 1984, 79, 228; H.

Pan-Hou and Y. Suda, Brain Res., 1987, 418, 198; T. Nishizaki and Y. Okada, ibid., 1988, 452,

11; H. Jackson and P. N. R. Usherwood, Trends Neurosci., 1988, 11, 278.

4 N. Kawai, A. Miwa, and T. Abe, Brain Res., 1982, 247, 169; T. Abe, N. Kawai, and A. Miwa, J.

Physiol., 1983, 339, 243; N. Kawai, S. Yamagishi, M. Saito, and K. Furuya, Brain Res., 1983,

278, 346; A. Miwa, N. Kawai, M. Saito, H. Pan-Hou, and M. Yoshioka, J. Neurophsiol., 1987,

58, 319.

5 M. Miyashita, H. Sato, A. Yoshikoshi, T. Told, M. Matsushita, H. Irie, T. Yanami, Y. Kikuchi, C.

Takasaki, and T. Nakajima, Tetrahedron Lett., 1992, 33, 2833.

6 M. Miyashita, H. Sato, M. Matsushita, Y. Kusumegi, T. Told, A. Yoshikoshi, T. Yanami, Y.

Kikuchi, C. Takasaki, T. Nakajima, and H. Irie, Tetrahedron Left., 1992, 33, 2837.

7 M. Miyashita, M. Matsushita, H. Sato, T. Told, T. Nakajima, and H. Irie, Chem. Lett., 1993, 929.

8 Other syntheses of spider toxins: NSTX-3: T. Teshima, T. Wakamiya, Y. Aramaki, T. Nakajima, N.

Kawai, and T. Shiba, Tetrahedron Lett., 1987, 28, 3509; D. M. Nason, V. J. Jasys, P. R.

Kelbaugh, D. Phillips, N. A. Saccomano, and R. A. Volkrnan, ibid., 1989, 30, 2337; T. Teshima,

T. Matsumoto, M. Miyagawa, T. Wakamiya, T. Shiba, N. Narai, M. Yoshioka, and T. Nakajima,

Tetrahedron, 1990, 46, 3819; JSTX-3: Y. Hashimoto, Y. Endo, K. Shudo, Y. Aramaki, N.

Kawai, and T. Nakajima, Tetrahedron Lett., 1987, 28, 35t l ; Argiotoxin: T. L. Shih, J. R-

Sanchez, and H. Mrozik, ibid., 1987, 28, 6015; M. E. Adams, R. L. Carney, F. E. Enderlin, E. T.

Fu, M. A. Jarema, J. P. Li, C. A. Miller, D. A. Schooley, M. J. Shapiro, and V. J. Venema,

Biochem. Biophys. Res. Commun., 1987, 148, 678; V. J. Jasys, P. R. Kelbaugh, D. M. Nason,

D. Phillips, N. A. Saccomano, and R. A. Volkamann, Tetrahedron Lett., 1988, 29, 6223;

Clavamine: T. Teshima, T. Matsumoto, M. Miyagawa, T. Wakamiya, T. Shiba, N. Narai, M.

Yoshioka, T. Nakajima, and N. Kawai, ibid., 1990, 46, 3819; Agelenopsis aperta: V. J. Jasys,

P. R. Kelbaugh, D. M. Nason, D. Phillips, K. J. Rosnack, N. A. Saccomano, J. G. Stroh, and R.

A. Volkamann, J. Am. Chem. Soc., 1990, 112, 6696; NPTX-9 and 11: B. W. Bycroft, W. C.

Chan, N. D. Hone, S. Millington, andd I. A. Nash, J. Am. Chem. Sac., 1994, 116, 7415; FTX:

I. S. Blagbrough and E. Moya, Tetrahedron Lett., 1994, 35, 2057.

9 The succinimidyl ester 4 was prepared from the methyl ester 3 by hydrolysis with 1N NaOH in EtOH

followed by treatment with N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide (DCC) in

AcOEt in 82% overall yield.

10 The product was purified by flash silica gel chromatography (CH2Cl2-acetone-EtOH = 4:1:0.5).

11 1H-NMR spectrum and [c¢] D value of natural NPTX-7 have not been measured yet owing to its

limited quantity.

(Received in Japan 16 March 1995; revised 10 May 1995; accepted 23 May 1995)