The first synthesis of clausenamine-A and cytotoxic activities of three biscarbazole analogues against cancer cells

Article abstract of DOI:10.1016/S0960-894X(00)00158-X

Clausemine-A (3), isolated from the stem and root bark of Clausena excavata, was synthesized using Suzuki cross-coupling and Oxidative coupling as the key step. Compound 3, and the other two structurally related biscarbazoles 1 and 2, showed potent cytotoxic activities a variety of human cancer cell lines in vitro. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00158-X

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1021±1023
The First Synthesis of Clausenamine-A and Cytotoxic Activities
of Three Biscarbazole Analogues Against Cancer Cells
Aimin Zhang and Guoqiang Lin*
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Lu, Shanghai, 200032, China
Received 5 January 2000; accepted 28 February 2000
AbstractÐClausemine-A (3), isolated from the stem and root bark of Clausena excavata, was synthesized using Suzuki cross-cou-
pling and Oxidative coupling as the key step. Compound 3, and the other two structurally related biscarbazoles 1 and 2, showed
potent cytotoxic activities against a variety of human cancer cell lines in vitro. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
6-amino-m-cresol (5) was employed, as depicted in
Scheme 1. There are three major stages involved in this
approach: (i) Suzuki cross-coupling of boronic acid
moiety and bromination moiety (7+8); (ii) regioselec-
tive bromination of the coupling product (9!10); (iii)
palladium(0) mediated cyclization of amino bromide
moiety (10!11).
Several biscarbazoles with diverse biological activities
have been isolated from di€erent natural sources over
the past decades.1 For example, dimeric O-demethyl-
murrayafoline A (1) was found to exhibit anti-
plasmodial activity against P. falciparum in vitro.2
Recently, clausenamine-A (3)3 was isolated from the
stem and root bark of Clausena excavata, which is used
as folk medicine for detoxication caused by the poiso-
nous snakebite in China. We4 have synthesized two
optically pure biscarbazoles through oxidative coupling
of phenolic monomer. As a continuation of our work, we
report herein on the ®rst synthesis of clausenamine-A (3)
and its cytotoxic activity. The activities of structurally
related analogues 1 and 2 were also described.
After trying to use several protected groups, tosylate (6)5
was employed as the electron-withdrawing group
(Scheme 2). Compound 7 was gained through electro-
philic aromatic bromination6 of 6 using bromodi-
methylsulfonium bromide generated in situ. The
brominate position substituted in the ortho position of
the amino group was con®rmed by NOE e€ects (94%
yield). The required biphenyl (9) is prepared quantita-
tively by Suzuki cross coupling of 3,5-dimethoxyphenyl
boronic acid (8)7 and 7 with 5% Pd(PPh3)4, 2 N aque-
ous Na2CO3 in benzene.8 Analysis of the electron dis-
tributing of 9 through the semiempirical MO calculation
using the hyperchem programs disclosed that the phenyl
owned two methoxy groups is electron abundant, which
®ts for the electrophilic bromination procedure. Bromi-
nation of 9 using 48% HBr in DMSO a€orded com-
pound 10 in 79.5% yield.6 The ®ve-membered ring
closure was successfully performed through a palla-
dium(0) mediated cyclization9 of amino bromide 10
which provided 9-H-carbazole 11 in 97% yield. Depro-
tection of tosylate group gave the monomer 4. Oxidative
coupling of the phenolic monomer 4 was completed by
aerial treatment of 4 with (t-BuO)2 in chlorobenzene to
provide the racemic clausenamine-A (3) in 90% yield.10
The structure of 3 was fully characterized by spectro-
Synthesis
To achieve a general and regiospeci®c synthesis of clau-
senamine-A (3), previously we chose the similar manner
as in the preparation of 1 and 2, which involved the
oxidative coupling of phenolic monomer. However,
owing to the electron-donating e€ect of the two meth-
oxy groups in the benzene ring, we failed to get the
desired coupling product through Pd(OAc)2 catalyzed
cyclization of N-(2,4-dimethoxy)-phenyl-2-benzyloxy-4-
methylaniline and cyclization of 4-methyl-cyclohexane-
1,2-dione-1-phenylhydrazone.4 Therefore, another syn-
thetic route starting from the commercially available
1
scopic analysis such as HNMR, IR, MS, and so on.11
*Corresponding author. Tel.: +86-21-64163300; fax: +86-21-64166263.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00158-X

1022
A. Zhang, G. Lin / Bioorg. Med. Chem. Lett. 10 (2000) 1021±1023
Scheme 1.
Scheme 2. Reagents and conditions: (i) TsCl, Et3N, CH2Cl2, rt, 84%; (ii) 48% HBr, DMSO, rt, 94%; (iii) 5% Pd(PPh3)4, 2 N Na2CO3, benzene, 3,5-
dimethoxyphenyl boronic acid, ethanol, re¯ux, 99%; (iv) 48% HBr, DMSO, rt, 79.5%; (v) 1.2 equiv Pd(PPh3)4, Na2CO3, toluene, re¯ux, 97%; (vi)
KOH, H2O, EtOH, re¯ux, 88%; (vii) (t-BuO)2, chlorobenzene, re¯ux, 90%.
Cytotoxicity assays
Table 1. The selected cancer cell growth inhibitory activity of com-
pounds 1±3 in vitro (GI50 values in mM)
With clausenamine-A (3) and its analogues 1 and 24 at
Cell linesa
1b
2 b
3
hand, their cytotoxic activities against cancer cells were
evaluated in the National Cancer Institute (USA) for the
disease-oriented antitumor screen in vitro,12 which deter-
mines a test agent's e€ect growth parameters against a
panel of approximately 60 human tumor cell lines. The
cytotoxic e€ects of 3 and the related compounds 1 and 2
are obtained as TGI or GI50 values, which represent the
molar drug concentration required to cause total growth
inhibition and half growth inhibition, respectively. The
results are presented in Table 1.
HL-60
K-562
MOLT-4
RPMI-8266
SR
NIC-H23
HCT-15
HT29
32.9
12.3
3.62
3.85
3.16
12.4
10.1
6.04
17.4
16.5
15.0
15.9
14.7
13.0
11.2
15.0
14.7
0.0321
4.04
3.23
3.61
3.97
4.48
4.20
3.71
5.11
4.34
3.82
4.78
4.27
3.78
4.49
4.34
4.50
2.82
9.70
8.59
10.5
9.01
8.20
8.28
8.03
SF-295
10.2
SK-MEL-5
OVCAR-5
A-498
CAKI-1
UO-31
MCF-7
8.38
9.03
7.82
9.89
6.84
8.68
As shown in Table 1, these biscarbazoles exhibited from
strong to moderate cytotoxic activities against a variety
of human cancer cell lines. Compound 2 showed strong
cytotoxic e€ects against leukemia cell lines, including
HL-60 (0.032 mM), while O-demethylmurrayafoline A
(1) has less activity compared with compounds 2 and 3.
In the MOLT-4, RPMI-8266 and SR assay, the GI50 of
compound 1 is better than the other two. Clausenamine-
A (3), which has two methoxy groups at the 6,60,8,80
position, proved to be the moderate active compound in
MDA-MB-435
MDA-N
12.0
8.83
aHl-60, K-562, MOLT-4, RPMI-8266, SR, leukemia cell lines; NIC-
H23, nonsmall cell lung cancer; HCT-15, HT29, colon cancer cell
lines; SF-295, CNS; SK-MEL-5, melanoma; OVCAR-5, A-498,
CAKI-1 and UO-31, ovarian and renal cancer cell lines; MCF-7,
MDA-MB-435 and MDA-N, breast cancer cell lines.
bThe spectroscopic data of compound 1 and 2 see ref 4.

A. Zhang, G. Lin / Bioorg. Med. Chem. Lett. 10 (2000) 1021±1023
1023
this study. All these biscarbazoles showed fairly good
activities against leukemia, nonsmall cell lung, colon,
CNS, melanoma, ovarian, renal, prostate, and breast
cancer.
2. Bringmann, G.; Ledermann, A.; Holenz, J.; Kao, M.-T.;
Busse, U.; Wu, H.-G.; Francois, G. Planta. Med. 1998, 64, 54.
3. Wu, T.-S.; Humg, S.-C.; Wu, P.-L. Tetrahedron. Lett. 1996,
37, 7819.
4. Lin, G.-Q.; Zhang, A.-M. Tetrahedron. Lett. 1999, 40, 341.
5. Kurita, K. Chem. Ind. (London) 1974, 345.
6. Majetich, G.; Hicks, R.; Reister, S. J. Org. Chem. 1997, 62,
4321.
7. Dol, G. C.; Kamer, P. C.; Leeuwen, P. W. N. M. Eur. J.
Org. Chem. 8, 359.
In summary, for the ®rst time we have accomplished the
preparation of clausenamine-A (3). Clausemanine-A
and its analogues exhibited important cytotoxic activ-
ities against a variety of human cancer cell lines in vitro.
8. (a) Miller, R. B.; Dugar, S. Tetrahedron. Lett. 1989, 30, 297.
(b) Miller, R. B.; Dugar, S. Organometallics 1984, 3, 1261.
9. (a) Boger, D. L.; Panek, J. S. Tetrahedron. Lett. 1984, 25,
3175. (b) Wood, J. L.; Stoltz, B. M.; Dietrich, H. J.; P¯um, D.
A.; Petsh, D. T. J. Am. Chem. Soc. 1997, 119, 9647.
10. Bringmann, G.; Ledermann, A.; Francios, G. Heterocycles
1995, 40, 293.
11. The spectroscopic data of compound 3: 1H NMR (300
MHz, CDCl3) 8.69 (s, 2H,-NH), 7.60 (s, 2H, 4-H), 7.04 (d,
J=1.68 Hz, 2H, 5-H), 6.45 (d, J=1.64 Hz, 2H, 7-H), 5.73 (s,
2H, -OH), 3.91 (s, 6H, -OCH3), 3.63 ( s, 6H, -OCH3), 2.15 (s,
6H, -CH3). FT±IR (KBr): 3398, 2932, 1595 cm 1. MS m/z (EI,
70 EV): 512(100), 497, 482, 256. HRMS calcd for C30H28N2O6
(M+): 512.1947, found 512.1970.
Acknowledgements
We are grateful to the National Science Foundation of
China (29772052) and the Chinese Academy Science
(KT951-A1-506-05) for ®nancial support of this work.
We also thank V. L. Narayanan and E. D. Sauaville for
the anticancer evaluation of compounds 1±3 at NIH.
References and Notes
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(b) Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simo,
R. M.; Tosini, S.; Skehan, P.; Scudiero, P. A.; Monks, A.;
Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1113.
1. (a) Wu, T.-S., Ohta, T.; Furukawa, H. Heterocycles 1983,
20, 1267. (b) Furukawa, H.; Wu, T.-S.; Ohata, T.; Kuoh, C.-S.
Chem. Pharm. Bull. 1985, 33, 4132.