Synthesis and cytotoxic activities of 1-benzylidine substituted β-carboline derivatives

Article abstract of DOI:10.1016/j.bmcl.2008.10.043

A series of new β-carboline derivatives, bearing a benzylidine substituent at position-1, has been prepared and evaluated in vitro against a panel of human cell lines. The N²-benzylated β-carbolinium bromates represented the most interesting cytotoxic activities. In particular, compounds 19 were found to be the most potent compounds with IC₅₀ values lower than 5 μM against 10 strains human tumor cell lines. These results confirmed that the N²-benzyl substituent on the β-carboline ring played an important role in the modulation of the cytotoxic activities and suggested that further development of such compounds may be interest.

Full text of DOI:10.1016/j.bmcl.2008.10.043

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6558–6561
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and cytotoxic activities of 1-benzylidine substituted
b-carboline derivatives
a
b
b
b
Rihui Cao a, , Wei Yi , Qifeng Wu , Xiangdong Guan , Manxiu Feng ,
*
Chunming Ma a, Zhiyong Chen a, Huacan Song a, Wenlie Peng b,
*
a School of Chemistry and Chemical Engineering, Sun Yat-sen (Zhongshan) University, 135 Xin Gang Xi Road, Guangzhou 510275, PR China
b School of Life Sciences, Sun Yat-sen (Zhongshan) University, 135 Xin Gang Xi Road, Guangzhou 510275, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new b-carboline derivatives, bearing a benzylidine substituent at position-1, has been pre-
pared and evaluated in vitro against a panel of human cell lines. The N2-benzylated b-carbolinium bro-
mates represented the most interesting cytotoxic activities. In particular, compounds 19 were found to
Received 20 May 2008
Revised 21 September 2008
Accepted 10 October 2008
Available online 14 October 2008
be the most potent compounds with IC50 values lower than 5 lM against 10 strains human tumor cell
lines. These results confirmed that the N2-benzyl substituent on the b-carboline ring played an important
role in the modulation of the cytotoxic activities and suggested that further development of such com-
pounds may be interest.
Keywords:
Harmine
b-Carbolines
Ó 2008 Elsevier Ltd. All rights reserved.
Cytotoxic activities
Structure–activity relationships
The b-carboline core is common to many natural and synthetic
products associated with a broad spectrum of biochemical effects
and pharmaceutical properties.1 Previously, numerous investiga-
tions focused on the effects of b-carboline alkaloids on the central
nervous system (CNS), such as their affinity with benzodiazepine
(BZ),2 5-hydroxytryptamine (5-HT),3 dopamine (DA),4 and imidaz-
oline5 receptors. However, considerable recent interests in these
alkaloids were stimulated by their potential antitumor activities.
Ishida et al.6 reported that harmine (Fig. 1), which naturally occurs
in the medicinal plants of Peganum harmala and Eurycoma longifo-
lia, and b-carboline analogues exhibited significant antitumor
vivo.10,12 The structure–activity relationships (SARs) analysis pro-
vided evidence that (1) the molecular feature essential for the anti-
tumor activity was the b-carboline moiety; (2) the introduction of
appropriate substituents into position-1, 2, 3 and 9 of b-carboline
ring remarkably enhanced the antitumor activities; (3) the meth-
oxy group at position-7 of b-carboline nucleus played a very crucial
role in determining their remarkable neurotoxic effects.1 Taking
advantage of previously developed SARs of b-carbolines as poten-
tial antitumor agents, in the present investigation we have de-
signed and synthesized a number of new b-carboline derivatives
bearing various 4-substituted benzylidine at position-1. The pur-
pose of this study was to investigate effect of benzylidine substitu-
ent on the antitumor activity, with the ultimate aim of developing
novel potent antitumor agents, together with lower side effects.
activities in vitro and a-(4-nitrobenzylidine)-harmine (Fig. 1) was
found to be the most active compound with a broad cytotoxicity
spectrum. b-Carbolines bearing a flexible alkylamine side chain
at position-3 demonstrated potent DNA intercalating abilities
resulting in remarkable antitumor activities.7 The complex polycy-
clic ring system in manzamine A can be replaced with simpler
amino substituents to provide active compounds.8 In addition, b-
carboline amino acid ester conjugates displayed potent cytotoxic
activities and the Lys/Arg conjugates demonstrated the most sig-
nificant antitumor activities in vitro.9 Our previous reports de-
scribed the syntheses of numerous b-carboline derivatives
bearing various substituents at position-1, 2, 3 and 9 of b-carboline
nucleus and evaluated their antitumor activities in vitro10–13 and in
N
N
H3CO
H3CO
N
H
N
H
CH3
HC
Harmine
NO2
α-(4-nitrobenzylidine)-harmine
* Corresponding authors. Tel.: +86 20 84110918; fax: +86 20 84112245.
E-mail addresses: caorihui@mail.sysu.edu.cn (R. Cao), pengwl@mail.sysu.edu.cn
(W. Peng).
Figure 1. Chemical structure of harmine and
a
-(4-nitrobenzylidine)-harmine.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.043

R. Cao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6558–6561
6559
DMF/NaH
N
N
H3CO
H3CO
N
CH3
N
H
CH2CH2CH2Br
CH3
(CH2)3
1
2
N
DMF/NaH
n-C4H9I
C4H9O
N
Acetic acid/HBr
HO
N
CH3
N
CH3
(CH2)3
(CH2)3
4
3
Scheme 1. Synthesis of 7-methoxy-9-(3-phenylpropyl)-1-methyl-b-carboline.
(CH3CO)2O
R1CHO
N
CH=CH R1
R2
N
CH3
R2
N
N
R3
R3
8
9
R1=C6H5 R2=H R3=H
4
5
6
7
R2=OC4H9 R3=(CH2)3C6H5
R2=H R3=H
R1=C6H4(p-OCH3) R2=H R3=H
10 R1=C6H2(3,4,5-trimethoxy) R2=H R3=H
11 R1=C6H4(p-OCH3) R2=H R3=n-C4H9
R2=H R3=n-C4H9
R2=H R3=(CH2)3C6H5
12 R1=C6H4(p-OCH3) R2=H R3=(CH2)3C6H5
13 R1=C6H4(p-OCH3) R2=OC4H9 R3=(CH2)3C6H5
14 R1=C6H4(p-NO2) R2=H R3=COCH3
15 R1=C6H4(p-NO2) R2=H R3=(CH2)3C6H5
16 R1=C6H4(p-NO2) R2=OC4H9 R3=(CH2)3C6H5
Br_
CH2Br
N+ CH2
R2
EA
N
CH=CH R1
R3
17 R1=C6H4(p-OCH3) R2=H
18 R1=C6H4(p-OCH3) R2=H
19 R1=C6H4(p-OCH3) R2=H
R3=H
R3=n-C4H9
R3=(CH2)3C6H5
20 R1=C6H4(p-OCH3) R2=OC4H9 R3=(CH2)3C6H5
21 R1=C6H4(p-NO2)
22 R1=C6H4(p-NO2)
R2=H
R3=COCH3
R2=OC4H9 R3=(CH2)3C6H5
Scheme 2. Synthesis of 1-benzylidene substituted b-carboline derivatives.
The synthesis of b-carbolines 2 and 5–7 has been described in
our previous reports.10,12 Compound 2 was demethylated in acetic
acid and hydrobomic acid (1:2) to afford the expected product 3
(yield 80%). O7-Butylated b-carboline 4 was prepared from com-
pound 3 by the action of sodium hydride in dry DMF followed by
addition of n-butyl iodide (Scheme 1).
carboline 14. The N2-benzylated b-carbolinium bromate deriva-
tives 17–2215 were prepared from compounds 9, 11–14, and 16
by the addition of benzyl bromide in refluxing ethyl acetate. How-
ever, the same synthetic procedure was used for the preparation of
N2-benzylated 15 but failed to afford the expected b-carbolinium
bromate.
Compounds 8–22 were prepared as shown in Scheme 2. 1-Ben-
zylidine substituted b-carbolines 8–1014 were readily prepared by
reaction of 1-methyl-b-carboline 5 with the corresponding aro-
matic aldehyde in refluxing acetic anhydride. The same synthetic
procedure was used for the preparation of compounds 11–16.14
Unexpectedly, the reaction of 1-methyl-b-carboline 5 with 4-nitro-
benzaldehyde in refluxing acetic anhydride gave N9-acetylated b-
The cytotoxic potential of all newly synthesized b-carboline
derivatives was evaluated in vitro against a panel of human tumor
cell lines according to procedures described in our previous re-
ports.10 The tumor cell line panel consisted of cervical carcinoma
(HeLa), liver carcinoma (Bel-7402 and HepG2), gastric carcinoma
(BGC-823), non-small cell lung carcinoma (A549), malignant mela-
noma (A375), renal carcinoma (786-0 and 769-P), colon carcinoma

6560
R. Cao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6558–6561
Table 1
Cytotoxic activities of b-carboline derivatives in vitroc (IC50 Ma)
, l
Compound
HeLa
Bel-7402
BGC-823
HepG2
A549
A375
786-0
HT-29
769-P
KB
5b
6b
7b
8
186
102
151
145
449
190
118
34.6
29.7
>200
>200
>200
>200
>200
22.5
7.7
215
264
987
>200
167
115
145
449
171
60.5
32.2
66.0
186
92.9
197
>200
>200
9.6
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
7.8
25.6
5.7
25.2
123
62.9
>200
>200
>200
4.9
5.1
4.6
8.2
19.8
3.1
—
—
—
>1000
139
142
50.5
88.8
>200
92.3
>200
>200
>200
32.1
6.7
36.7
47.1
12.8
38.1
127
78.6
>200
>200
>200
10.8
9.1
71.4
42.2
23.9
59.5
>200
64.3
>200
>200
>200
28.2
8.4
37.5
12.9
14.4
67.4
92.3
89.1
>200
>200
>200
10.0
11.2
3.2
62.9
21.3
27.8
>200
>200
54.3
>200
>200
>200
9.4
7.8
4.3
3.2
10.5
2.5
>200
29.5
24.6
30.1
58.9
150
>200
>200
>200
17.9
3.9
9
10
11
12
13
14
15
16
17
18
19
20
21
22
66.2
141
>200
>200
>200
>200
>200
22.9
21.8
0.46
7.1
6.7
2.1
8.9
5.2
4.5
6.8
14.4
2.9
2.9
1.6
17.9
1.5
3.9
3.4
7.6
2.8
0.68
7.3
25.3
4.5
0.93
2.7
15.4
2.4
4.6
28.6
2.7
14.8
4.1
1.3
a
Cytotoxicity as IC50 for each cell line, is the concentration of compound which reduced by 50% the optical density of treated cells with respect to untreated cells using the
MTT assay.
b
c
See Ref. 8.
Data represent the mean values of three independent determinations.
(HT-29), and oral squamous carcinoma (KB). Compounds 8–16
were all prepared in the form of hydrochloride in order to enhance
the solubility in aqueous solution by the usual methods before use.
The results were summarized in Table 1.
In conclusion, a number of novel b-carboline derivatives de-
scribed in this paper proved to be remarkably potent antitumor
activities. In comparison with already published antitumor agents
of similar chemical structure, N2-benzylated b-carbolinium bro-
mates 19, 20, and 22 were found to be the most potent compounds
As shown in Table 1, compounds 8–10 with no substituents at
position-9 exhibited moderate cytotoxic activities, but compound
10, bearing a 3,4,5-trimethoxybenzylidine substituent at posi-
tion-1, was more active. In our previous investigation, we found
that introducing an n-butyl or phenylpropyl substituent into posi-
tion-9 of b-carboline nucleus facilitated antitumor activities in vitro
with IC50 values lower than 10 lM against a panel of human tumor
cell lines. Therefore, N2-benzylated b-carbolinium bromates 19, 20,
and 22 can be considered promising leads for further structural
modifications guided by the valuable information derivable from
our detailed SARs.
10–13
and in vivo.10,12 Unfortunately, in the present investigation,
compounds 12–13 and 15–16 were almost inactive to all tumor
Acknowledgment
cell lines investigated at the concentration of 200 lM.
The N2-benzylated b-carboline derivatives 17–22 represented
Authors are thankful to Xinjiang Huashidan Pharmaceutical Co.
Ltd for financial support.
the most interesting cytotoxic activities. As predicted, the IC50 val-
ues of these compounds were lower than 10 lM against most of
human tumor cell lines investigated. Interestingly, compounds 19
References and notes
was found to be the most active derivative with IC50 value of
0.46 lM, 0.68 lM, and 0.93 lM against BGC-823, A375, and KB cell
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lines, respectively. These results further confirmed that the posi-
tion of the N2-benzylated substituent on the b-carboline ring
played a very vital role in the modulation of the cytotoxic
activities.
Some important molecular mechanisms of action of this class of
bioactive compounds have been recently reviewed.1 However, by
far, the underlying mechanism of action for the antitumor effects
of b-carbolines has not been completely defined. Our previous
investigations reported that the ability of b-carbolines to act as
intercalating agents and Topo I inhibitors was related to the antitu-
mor activity16 and b-carboline derivatives can pass through cell
membrane and penetrate into nucleus quickly resulting in interca-
lating into DNA in cells.17 Very recent researches in our laboratory
indicated that N2-benzylated b-carbolinium ions can penetrate into
plasma membrane and nuclear envelop more easily than those
molecules without substituents at position-2. However, further
studies will be needed to elucidate the underlying mechanism of
action of these compounds completely.
4. Abdel-Fattah, A. F. M.; Matsumoto, K.; Gammaz, H. A. F.; Watanabe, H.
Pharmacol. Biochem. Behav. 1995, 52, 421.
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9. Zhao, M.; Bi, L.; Wang, W.; Wang, C.; Baudy-Floc’h, M.; Ju, J.; Peng, S. Bioorg.
Med. Chem. 2006, 14, 6998.
10. Cao, R.; Chen, Q.; Hou, X.; Chen, H.; Guan, H.; Ma, Y.; Peng, W.; Xu, A. Bioorg.
Med. Chem. 2004, 12, 4613.
Furthermore, in order to confirm the utility of the b-carbolini-
um ion derivatives as an interesting antitumor agent, compounds
18–20 and 22 are now selected and submitted to further acute tox-
icity and antitumor activity studies in animal models, and the rel-
ative possible results will be reported in due course.

R. Cao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6558–6561
6561
11. Cao, R.; Peng, W.; Chen, H.; Hou, X.; Guan, H.; Chen, Q.; Ma, Y.; Xu, A. Eur. J.
Med. Chem. 2005, 40, 249.
12. Cao, R.; Chen, H.; Peng, W.; Ma, Y.; Hou, X.; Guan, H.; Liu, X.; Xu, A. Eur. J. Med.
Chem. 2005, 40, 991.
13. Guan, H.; Chen, H.; Peng, W.; Ma, Y.; Cao, R.; Liu, X.; Xu, A. Eur. J. Med. Chem.
2006, 41, 1167.
8.46–8.47 (1H, d, J = 5.0 Hz, H-3); 8.10–8.12 (1H, d, J = 7.5 Hz, H-4); 7.66–7.86
(3H, m, H-5, H-6, H-7); 7.53–7.55 (3H, m, H-8, PhH); 7.15–7.33 (7H, m, PhH);
6.94–6.95 (2H, d, J = 8.5 Hz, –CH@CH–); 4.58–4.61 (2H, m, NCH2CH2CH2Ph);
3.86 (3H, s, OCH3); 2.75–2.78 (2H, m, NCH2CH2CH2Ph); 2.30–2.33 (2H, m,
NCH2CH2CH2Ph). Anal. Calcd for C29H26N2O: C, 83.22; H, 6.26; N, 6.69. Found:
C, 83.40; H, 6.29; N, 6.67.
14. General procedure for the preparation of 1-benzylidine b-carboline derivatives 8–
16. A mixture of 4–7 (10 mmol), acetic anhydride (50 ml) and aldehydes (50–
100 mmol) was refluxed for 24–48 h. After completion of the reaction as
indicated by TLC, the solution was poured into ice-water (200 ml) and made
basic with sodium bicarbonate. The aqueous mixture was extracted with ethyl
acetate, and the organic phase was washed with water and brine and then
dried over anhydrous sodium sulfate. The solvent was removed under vacuum,
and the residue was dissolved in ethanol and made acidic with concentrated
hydrochloric acid. The solvent was evaporated in reduced pressure and the
resulting oil was crystallized from acetone to give yellow solid. The solid was
dissolved in water and made basic with sodium bicarbonate, and the aqueous
mixture was extracted with ethyl acetate. The organic phase was washed with
water and brine, dried over anhydrous sodium sulfate, concentrated under
vacuum. The oil residue was crystallized to give 8–16 in 12–65% yields.
Compound 12: yield 53%, mp 136–137 °C. FAB-MS m/e (M+1) 419. IR (KBr)
3053, 3030, 2970, 2922, 2841, 1630, 1603, 1555, 1509, 1446, 1415, 1361, 1328,
15. General procedure for the preparation of 1-benzylidine b-carbolinium bromates
17–22.
A mixture of 9, 11–14 or 16 (2 mmol) and benzyl bromide (10–
20 mmol) in ethyl acetate (50 ml) was refluxed for 5–10 h. After completion of
the reaction as indicated by TLC, the solution was cooled and filtered and
crystallized to afford 17–22 in 44–76% yields. Compound 19: yield 60%, mp
207–209 °C. FAB-MS m/e 509. IR (KBr) 3412, 3029, 3002, 2936, 2837, 1622,
1606, 1515, 1457, 1339, 1256, 1179, 1033, 971, 820 cmÀ1 1H NMR (500 MHz,
.
DMSO-d6) d 8.98–8.99 (1H, d, J = 6.5 Hz, H-3); 8.89–.90 (1H, d, J = 6.5 Hz, H-4);
8.57–.59 (1H, d, J = 8.5 Hz, H-5); 7.88–7.93 (2H, m, H-6, H-7); 7.03–7.04 (15H,
m, H-8, PhH); 6.82–6.84 (2H, d, J = 8.5 Hz, –CH@CH–); 6.07 (2H, s, CH2Ph);
4.59–.62 (2H, t, NCH2CH2CH2Ph); 3.84 (3H, s, OCH3); 2.49–.51 (2H, m,
NCH2CH2CH2Ph); 2.28–.30 (2H, m, NCH2CH2CH2Ph). Anal. Calcd for
C36H33BrN2O: C, 73.34; H, 5.64; N, 4.75. Found: C, 73.20; H, 5.66; N, 4.78.
16. Cao, R.; Peng, W.; Chen, H.; Ma, Y.; Liu, X.; Hou, X.; Guan, H. Biochem. Biophys.
Res. Commun. 2005, 338, 1157.
17. Guan, H.; Liu, X.; Peng, W.; Cao, R.; Ma, Y.; Chen, H.; Xu, A. Biochem. Biophys.
Res. Commun. 2005, 342, 894.
1291, 1242, 1222, 1171, 1032, 966, 821 cmÀ1
.
1H NMR (500 MHz, CDCl3) d