Synthesis and biological evaluation of N9-substituted harmine derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2016.06.087

A series of N⁹-substituted harmine derivatives were synthesized and evaluated for their anticancer activity on a panel of cancer cell lines, their apoptosis induction and their cell cycle effects. The results showed that N⁹-substituted harmine derivatives had anticancer effects. In particular, N⁹-haloalkyl derivatives 9a–9c and N⁹-acyl harmine derivatives 11c and 11d, with IC₅₀values less than 1?μM, were more potent than doxorubicin against A-549 and/or MCF-7 cell lines. Moreover, structure–activity relationships (SARs) indicated that introducing a haloalkyl or benzenesulfonyl group in the N⁹-position of harmine could significantly increase the anticancer activity. The most active compound (11d) caused cell cycle arrest in the G2/M phase, and induced cell apoptosis in a dose-dependent manner.

Full text of DOI:10.1016/j.bmcl.2016.06.087

Accepted Manuscript
9
Synthesis and Biological Evaluation of N -substituted Harmine Derivatives as
Potential Anticancer Agents
Hongtao Du, Shan Tian, Juncheng Chen, Hongling Gu, Na Li, Junru Wang
PII:
S0960-894X(16)30698-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.06.087
BMCL 24040
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
24 February 2016
17 June 2016
29 June 2016
Please cite this article as: Du, H., Tian, S., Chen, J., Gu, H., Li, N., Wang, J., Synthesis and Biological Evaluation
9
of N -substituted Harmine Derivatives as Potential Anticancer Agents, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.06.087
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

9
Synthesis and Biological Evaluation of N -substituted Harmine
Derivatives as Potential Anticancer Agents
a, b
a
a
a
a
a,
Hongtao Du , Shan Tian , Juncheng Chen , Hongling Gu , Na Li , Junru Wang *
a
College of Science, Northwest A&F University, Yangling 712100, Shaanxi Province, P. R. China
b
College of Plant Protection, Northwest A&F University, Yangling 712100, Shaanxi Province, P. R.
China
*
Corresponding author (J.W). Tel.: +86-29-87092829; Fax: +86-29-87092829; Email:
wangjunru@nwsuaf.edu.cn (J.R. Wang).
E-mails: duht@nwsuaf.edu.cn (H.T. Du); stian@nwsuaf.edu.cn (S. Tian); cjc2009@nwsuaf.edu.cn
(
J.C.
Chen);
hlgu@nwsuaf.edu.cn
(H.L.
Gu);
lnuk@nwsuaf.edu.cn
(N.
Li);
wangjunru@nwsuaf.edu.cn (J.R. Wang)
ABSTRACT
9
A series of N -substituted harmine derivatives were synthesized and evaluated for their
anticancer activity on a panel of cancer cell lines, their apoptosis induction and their cell cycle
9
effects. The results showed that N -substituted harmine derivatives had anticancer effects. In
9
9
particular, N -haloalkyl derivatives 9a–9c and N -acyl harmine derivatives 11c and 11d, with IC50
values less than 1 μM, were more potent than doxorubicin against A-549 and/or MCF-7 cell lines.
Moreover, structure–activity relationships (SARs) indicated that introducing a haloalkyl or
9
benzenesulfonyl group in the N -position of harmine could significantly increase the anticancer
activity. The most active compound (11d) caused cell cycle arrest in the G2/M phase, and induced
cell apoptosis in a dose-dependent manner.
Keywords: Harmine derivatives; Anticancer activities; Structure–activity relationships (SARs);
1

Apoptosis; Cell cycle arrest
Cancer is one of main diseases that poses a direct threat to human health and life. Approximately
1
seven million people die from cancer every year, and this number is growing rapidly. Most
2
anticancer drugs inhibit the proliferation of cancer cells through inducing apoptosis. However, the
3
, 4
resistance of cancer cells to cytotoxic drugs can lead to treatment failure. Therefore, it is of utmost
importance to develop new drugs for cancer. Ingenious modification of anticancer compounds could
improve their properties and/or decrease their side effects, and is one of feasible ways to search for
5
new drugs.
6
Many compounds containing an indole or pyridine structure, such as HYL-6d (1) and E7010
7
(
2), have significant anti-proliferative activity (Fig. 1). Harmine, originally isolated from the seeds
of Peganum harmala in 1847, is a typical β-carboline alkaloid, having a core indole structure and a
8
pyridine ring, and is endowed with diverse pharmacological properties, including antiinflammatory,
9
10
11
antimalarial, anti-HIV, and anticancer activity. Previous investigations have shown that harmine
12–14
demonstrates remarkable anticancer potential both in vitro and in vivo.
this is that harmine-mediated inhibition of DYRK1A induces the activation of caspase-9,
leads to massive apoptosis in different human cell types. Some studies have also revealed that
One of the reasons for
1
5,16
which
9
17
introducing appropriate substituents in the N -position of β-carboline, such as in HAC-Y6 (3) and
1
4
9
-phenylpropyl-harmine (4), can augment antitumor activity (Fig. 1). However, the current
14
research have limitations, and some research groups have presented different views. Cao’s group
9
reported that a short alkyl or benzyl substituent at the N -position of harmine can increase the
1
8
anticancer activity significantly, while Ghasemi and Davoudian concluded that a medium-sized
2

aliphatic alkyl group is favored. In the present study, to improve the anticancer activity of harmine
derivatives and to illuminate their antitumor structure–activity relationships (SARs), various
9
N -substituted harmine derivatives were synthesized (Fig. 1), their anticancer activity evaluated in
vitro and a preliminary study made of the mechanism of action.
Fig. 1 Previous studies and design strategy of 9-substituted harmine derivatives
9
The synthetic route for obtaining harmine and its N -substituted derivatives is depicted in
Scheme 1. 1,2,3,4-Tetrahydro-harmine (6) was prepared from 6-methoxy-tryptamine (5) via a
19
Pictet–Spengler reaction according to a previously described method. Compound 6 was then
oxidized to produce the harmine (7) using Pd/C (5%) in refluxing toluene. Harmine (7) was
N-alkylated by treatment with sodium hydride (NaH) and an alkyl or benzyl halide in
dimethylformamide (DMF) at room temperature, to yield compounds 8a–8g, 9a–9d and 12a–12f.
9
The N -acyl harmine derivatives 10a, 10b and 11a–11d were synthesized by reaction with an acyl
chloride, such as acetyl chloride, benzoyl chloride or benzenesulfonyl chloride, in a mixture of
3

pyridine and DMF at room temperature. The chemical structures of all synthetic products were
1
13
confirmed by mass spectroscopy and H and C NMR spectroscopy.
Scheme 1. Synthesis of 9-substituted harmine derivatives. Reagents and conditions: (a) HCl (aq. 0.1N),
35% acetaldehyde, 40 °C, 6 h; (b) 5% Pb/C, dry toluene, reflux, 24 h; (c) NaH, dry DMF, RBr or RI, rt, 12
h; (d) NaH, dry DMF, RBr or RCl , rt, 8 h; (e) RCOCl or ArSO Cl, pyridine, DMF, rt, 2 h.
2
2
2
The in vitro anticancer activity of the synthesized compounds was evaluated against four human
2
0
cancer cell lines (MCF-7, SGC-7901, SMMC-7721 and A-549) by means of an MTT assay with
doxorubicin as a positive control. The IC50 values (the concentration of causing 50% inhibition of
4

9
cancer cell growth) are listed in Table 1. The results show that most N -substituted harmine
derivatives have moderate to excellent anticancer activity for the four human cancer lines tested.
Furthermore, all the synthesized compounds displayed much stronger activity against SGC-7901 and
9
A-549 cells than did the parent compound (7), which suggests that N -substituted harmine
derivatives could have a significantly improved the anticancer effect.
9
The N -alkyl derivatives 8a–8g were found to be more potent than compound 7 in the inhibition
of cancer cell growth. Of these compounds, 8g exhibited good activity against SGC-7901,
SMMC-7721 and A-549 with IC50 values of 6.68, 6.95 and 4.77 μM, respectively. Unfortunately,
there was no obvious pattern of the impact of alkyl chain length on inhibitory activity.
Table 1. In vitro cytotoxic activities of 9-substituted harmine derivatives against four human cancer cell lines
a
IC50 (μM)
9
Compounds
R
MCF-7
SGC-7901
SMMC-7721
A-549
8
8
8
8
8
8
8
9
9
9
9
1
1
1
1
1
1
a
Methyl
> 50
9.67±0.06
29.62±0.62
19.35±0.08
11.35±0.08
20.50±0.75
13.29±0.36
6.68±0.20
10.62±0.20
9.34±0.12
6.94±0.24
7.21±0.10
6.39±0.06
8.76±0.17
7.69±0.34
2.88±0.09
5.95±0.10
1.56±0.01
43.62±0.82
23.98±0.17
48.80±0.75
28.64±0.85
17.20±1.01
14.39±0.93
6.95±0.14
8.34±0.31
6.49±0.22
3.79±0.09
17.94±0.58
> 50
4.97±0.30
26.35±1.04
9.66±0.51
6.09±0.47
6.37±0.35
4.89±0.22
4.77±0.17
12.19±0.06
4.80±0.05
0.87±0.01
8.73±0.35
1.27±0.01
14.61±0.39
2.81±0.13
1.33±0.08
0.83±0.04
0.48±0.01
b
Ethyl
> 50
c
Propyl
> 50
d
Butyl
> 50
e
Pentyl
> 50
f
Hexyl
> 50
g
Octyl
> 50
a
4-Bromobutyl
5-Bromopentyl
6-Bromohexyl
4-Chlorobutyl
Acetyl
0.85±0.07
0.76±0.03
0.45±0.02
2.94±0.28
> 50
b
c
d
0a
0b
1a
1b
1c
1d
Dodecanoyl
Benzoyl
> 50
49.06
> 50
> 50
4-Methylbenzoyl
Benzenesulfonyl
4-Methylbenzenesulfonyl
> 50
> 50
> 50
> 50
> 50
> 50
5

1
1
1
1
1
1
2a
2b
2c
2d
2e
2f
Benzyl
>50
38.20±0.86
17.21±0.25
5.29±0.08
43.63±2.51
7.21±0.10
6.47±0.07
40.82±1.22
1.20±0.03
45.62±1.89
39.46±1.09
21.34±0.84
>50
7.49±0.11
8.34±0.16
3.89±0.02
5.67±0.06
8.73±0.35
18.39±0.12
42.25±2.17
0.76±0.02
2-Methylbenzyl
4-Methylbenzyl
2-Fluorobenzyl
4-Fluorobenzyl
3,5-Difluorobenzyl
H
>50
>50
>50
>50
>50
>50
>50
Harmine
Dox.
68.33±2.82
0.83±0.02
59.44±1.98
1.39±0.05
-
a
Human tumor cells were treated with six concentrations each for 48 h. IC50 values (the concentration of 50% proliferation-inhibitory
effect) were calculated by the Logit method from the results of at least three independent tests and expressed as the mean ± SD.
9
The N -haloalkyl derivatives 9a–9g showed moderate or potent anticancer activity against all
9
tested cells, and the N -bromobutyl derivative 9a (IC50=0.85–12.89 μM) was more potent than the
9
9
N -chlorobutyl compound 9d (IC50=2.94–17.94 μM). Among the N -bromoalkyl harmines 9a−9c,
compounds 9b and 9c were the most promising compounds against all tested cell lines, with IC50
9
values of 0.76–9.34 and 0.45–6.94 μM, respectively. Interestingly, all of these N -haloalkyl
9
molecules were more potent in inhibiting cancer cell lines growth than the corresponding N -alkyl
derivatives (the following pairs of compounds should be compared: 8d vs 9a, 8e vs 9b, and 8f vs 9c)
9
or harmine. The results indicate that introducing a haloalkyl group in the N -position is beneficial in
improving anticancer activity.
9
The dodecanoyl derivative (10b) was found to be less potent than the N -acetyl derivative (10a).
In the series of compounds having a benzyl, benzoyl or benzenesulfonyl substituent at the
9
9
N -position of the indole ring, the order of potency was found to be N -benzenesulfonyl >
9
9
N -benzoyl > N -benzyl (11a > 11c > 12a, and 11b > 11d > 12b). Compounds with a 4′-aryl
9
substituent at N showed lower anticancer activity than compounds having a 4′-methylaryl
substituent (11a < 11b, 11c < 11d, and 12a < 12b). Promisingly, compounds 11c and 11d were seen
to selectively inhibit the growth of A-549 cells, with IC50 values of 0.83 and 0.48 μM, respectively,
6

and were far more potent than the parent compound (7) (IC50 = 42.25 μM) and of equal potency to
doxorubicin (IC50 = 0.76 μM).
9
Based on the above analysis, some valuable SARs can be summarized (Fig. 2): (1) N -alkyl and
9
9
N -aryl groups are beneficial to anticancer activity; and (2) N -bromo alkyl derivatives are more
9
9
potent than chloro-substituted and N -alkyl derivatives, and for N -bromoalkyl derivatives the
9
inhibitory activity increases with increasing alkyl chain length; (3) in the series of N -aryl derivatives,
9
9
9
the activity sequence was N -benzenesulfonyl > N -benzoyl > N -benzyl. In addition, the
4
′
introduction of a methyl group in position C could significantly improve the anticancer activity.
Fig. 2 Structure-activity relationships (SARs) of 9-substituted harmine derivatives.
To clarify the effect of 9-substituted harmine derivatives on cell growth, the effect of compound
11d on cell-cycle progression in A-549 cells was investigated. A-549 cells were treated with 11d at
concentrations of 0.1, 0.5 and 1 μM for 48 h, and the cell-cycle distributions analyzed by flow
cytometry. As can be seen in Fig. 3, compound 11d at a concentration of 0.1 μM induced a slight
accumulation of cells in the G2/M phase (16.42%) compared with the control group (14.62%). When
7

the concentration of compound 11d was 0.5 μM, the percentage of cells in the G2/M phase was
markedly increased, from 14.62% to 31.09%. Meanwhile, the percentage of cells in the G0/G1 phase
was significantly decreased from 53.54% to 36.99%. When the A-549 cells were treated with
compound 11d at a concentration of 1 μM, the percentage of cells in the G2/M phase was
significantly increased to 36.64%. These results suggested that compound 11d could arrest A-549
cells in the G2/M phase in a dose-dependent manner. It can also be seen from Fig. 3 that compound
11d induced a significant and dose-dependent increase in the percentage of A-549 cells in the sub-G1
phase, which indicates that the A-549 cells treated with compound 11d had undergone apoptosis.
Therefore, the induction of apoptosis in A-549 cells by compound 11d was determined using an
2
1, 22
Annexin V-FITC/PI double staining assay
with 11d at concentrations of 0.1, 0.5 and 1 μM for 48 h, the rate of apoptosis of the cells was
1.44%, 54.29% and 71.70%, respectively, all of which are significantly higher than the rate
. As shown in Fig. 4, after treatment of the A-549 cells
2
achieved with blank control group (3.27%, DMSO). This finding indicates that 9-substituted harmine
derivatives could induce apoptosis of A-549 cells in a dose-dependent manner.
8

Fig. 3 Effects of 11d on the cell cycle in A-549 cells. The cells were harvested and analyzed for apoptosis by flow
cytometric analysis after treatment with 11d (0, 0.1, 0.5 and 1 μM) for 48 h.
Fig. 4 Effects of compound 11d on the induction of apoptosis in A-549 cells. The cells were treated with 11d (0, 0.1,
0.5 and 1 μM) for 48 h. The cells were then harvested and analyzed for cell-cycle progression by flow cytometry.
9

9
In summary, a series of N -substituted harmine derivatives were synthesized and their anticancer
activity against four human tumor cell lines in vitro evaluated. The majority of the derivatives
9
exhibited potent anticancer activity. SARs studies indicated that the N -position plays an important
role in modulating the anticancer activity, and that introducing a haloalkyl or benzenesulfonyl group
9
in the N -position is beneficial to anticancer activity. The preliminary study of the mechanism
revealed that compound 11d could cause cell cycle arrest in the G2/M phase and induce cell
apoptosis in a dose-dependent manner. In conclusion, compounds 9a–9c, 11c and 11d are good
candidates for further study and development as anticancer agents.
Acknowledgments
This work was supported by grants from the Program for Natural Science Foundation of China
(
Grant No. 31270388) and the Fundamental Research Funds for the Central Universities (Grant No.
QN2011066).
Supplementary data
Supplementary data (experimental procedure and spectroscopic characterizations of the
compounds) associated with this article can be found at XXXXXXXX.
References and notes
1
2
3
4
Varmus, H. Science. 2006, 312, 1162.
Fesik, S. W. Nat. Rev. Cancer. 2005, 5, 876.
Ali I.; Wani, W. A; Saleem, K.; Haque, A. Anticancer Agents Med. Chem. 2013, 13, 296.
Leite de Oliveira, R.; Deschoemaeker, S.; Henze, A.-T.; Debackere, K.; Finisguerra, V.; Takeda,
Y.; Roncal, C.; Dettori, D.; Tack, E.; Jönsson, Y. Cancer cell. 2012, 22, 263.
Gribble G. W.; Badenock, J. C.; Heterocyclic Scaffolds II: Reactions and Applications of
5
10

Indoles, Springer. 2010, 14.
6
7
8
9
1
1
1
1
1
1
1
Liu, C. H.; Lin, C.; Tsai, K. J.; Chuang, Y. C.; Huang, Y. L.; Lee, T. H.; Huang, L. J.; Chan, H.
C. Oncol. Rep. 2013, 29, 1501.
Yoshimatsu, K.; Yamaguchi, A.; Yoshino, H.; Koyanagi, N.; Kitoh, K. Cancer. Res. 1997, 57,
3
208.
Dighe, S. U.; Khan, S.; Soni, I.; Jain, P.; Shukla, S.; Yadav, R.; Sen, P.; Meeran, S. M.; Batra, S.
J. Med. Chem. 2015, 58, 3485.
Huang, H. B.; Yao, Y. L.; He, Z. X.; Yang, T. T.; Ma, J. Y.; Tian, X. P.; Li, Y. Y.; Huang, C. G.;
Chen, X. P.; Li, W. J.; Zhang, S.; Zhang, C. S.; Ju, J. H. J. Nat. Prod. 2011, 74, 2122.
0 Ashok, P.; Chander, S.; Balzarini, J.; Pannecouque C.; Murugesan, S. Bioorg. Med. Chem. Lett.
2
015, 25, 1232.
1 Chen, Y. F.; Kuo, P. C.; Chan, H. H.; Kuo, I. J.; Lin, F. W.; Su, C. R.; Yang, M. L.; Li, D. T.;
Wu, T. S.; J. Nat. Prod. 2010, 73, 1993.
2 Du, H. T.; Gu, H. L.;Li, N.; Wang, J. R. Med. Chem. Commun. 2016, DOI:
1
0.1039/c5md00581g.
3 Frederick, R.; Bruyere, C.; Vancraeynest, C.; Reniers, J.; Meinguet, C.; Pochet, L.; Backlund,
A.; Masereel, B.; Kiss, R.; Wouters, J. J. Med. Chem. 2012, 55, 6489.
4 Cao, R. H.; Chen, Q.; Hou, X. R.; Chen, H. S.; Guan, H. J.; Ma, Y.; Peng, W. L.; Xu, A. L.
Bioorgan. Med. Chem. 2004, 12, 4613.
5 Göckler, N.; Jofre, G.; Papadopoulos, C.; Soppa, U.; Tejedor, F. J.; Becker, W. FEBS. J. 2009,
2
76, 6324.
6 Seifert, A.; Allan, L. A.; Clarke, P. R. FEBS. J. 2008, 275, 6268.
11

1
7 Lin, Y. C.; Tsai, J. Y.; Yang, J. S.; Lee, Y. H.; Hamel, E.; Lee, K. H.; Kuo, S. C.; Huang, L. J.
Int. J. Oncol. 2013, 43, 1596.
1
1
8 Ghasemi, J. B.; Davoudian, V. J. Chem-Ny. 2014, 1.
9 Bi, W.; Bi, Y.; Xue, P.; Zhang, Y. R.; Gao, X.; Wang, Z. B.; Li, M.; Baudy-Floc'h, M.;
Ngerebara, N.; Li, X. X.; Gibson, K. M.; Bi, L. R. Eur. J. Med. Chem. 2011, 46, 2441.
0 Mosmann, T. J. Immunol. Methods. 1983, 65, 55.
2
2
1 van Engeland, M.; Nieland, L. J. W.; Ramaekers, F. C. S.; Schutte, B.; Reutelingsperger, C. P.
M. Cytometry. 1998, 31, 1.
2
2 Baloch, S. K.; Ma, L.; Wang, X. L.; Shi, J.; Zhu, Y.; Wu, F. Y.; Pang, Y. J.; Lu, G. H.; Qi, J. L.;
Wang, X. M. Rsc. Adv. 2015, 5, 31759.
12

Graphical Abstract