Synthesis and evaluation of nitrate derivatives of colchicine as anticancer agents

Article abstract of DOI:10.1016/j.cclet.2010.12.037

To search for more potent antitumor agent, a series of novel nitric oxide-donating colchicine (Col) derivatives (6a-f, 8a and b) were synthesized by coupling nitrates with N-methyl colchiceinamide. Their cytotoxicity against four human cancer cell lines in vitro were evaluated by MTT assay. It was found that many of the derivatives displayed significant activity, particularly, compounds 6c, 8a and 8b showed more potent cytotoxic activities than Col.

Full text of DOI:10.1016/j.cclet.2010.12.037

Available online at www.sciencedirect.com
Chinese Chemical Letters 22 (2011) 768–770
www.elsevier.com/locate/cclet
Synthesis and evaluation of nitrate derivatives
of colchicine as anticancer agents
a
a
b
c
Li Hong Shen a, , Ya Li , Da Hai Zhang , Yi Sheng Lai , Li Jie Liu
*
a Department of Chemistry, Handan College, Handan 056005, China
b Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, China
c Hebei Yiling Medicine Corporation Ltd., Shijiazhuang 050035, China
Received 29 September 2010
Available online 16 April 2011
Abstract
To search for more potent antitumor agent, a series of novel nitric oxide-donating colchicine (Col) derivatives (6a–f, 8a and b)
were synthesized by coupling nitrates with N-methyl colchiceinamide. Their cytotoxicity against four human cancer cell lines in
vitro were evaluated by MTTassay. It was found that many of the derivatives displayed significant activity, particularly, compounds
6c, 8a and 8b showed more potent cytotoxic activities than Col.
# 2010 Li Hong Shen. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Colchicine; N-methyl colchiceinamide; Nitrates; Cytotoxicity
Colchicine (Col, 1), the major alkaloid in Colchicum autumnale and Gloriosa superba is best known for its
antimitotic effects, and is a potent drug that interferes with microtubules both in vitro and in vivo, thereby causing cells
to accumulate in apparent mitotic arrest during the cell cycle [1,2]. Col has been used in the treatment of acute gout,
familial Mediterranean fever, scleroderma, amyloidosis, Behcet’s disease, liver disorder, tumor growth, particularly
cirrhosis [3–5]. Although Col is a potent antimitotic agent, its medicinal uses are limited due to its high toxicity. Much
interest has focused on structural modifications in the hope of improving the antitumor activity [6]. N-methyl
colchiceinamide (2, Scheme 1), a synthetic derivative of Col, showed considerably higher stability toward acid
hydrolysis and is a slightly less active antitumor and toxicity agent than Col [3].
Nitric oxide (NO) is a ubiquitous free radical in cells and tissues. It is produced in the body by an enzyme called the
NO synthases (NOS)[0], which converts the amino acid L-arginine to NO and L-citrulline. NO displays diverse potent
physiological actions such as vascular relaxation, neurotransmission, and events of the immune system. NO can also
be generated from synthetic NO-releasing agents, such as nitrate, furoxan, S-nitrosothiol, and diazeniumdiolate [7,8].
Studies showed that high levels of NO were cytotoxic and could induce the apoptosis of tumor cells [9]. In addition, it
was reported that NO conjugates of certain cytotoxic agents showed selective cytotoxicity and could be developed as
potential antitumor drugs [10,11].
* Corresponding author.
E-mail address: latishen@sohu.com (L.H. Shen).
1001-8417/$ – see front matter # 2010 Li Hong Shen. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2010.12.037

[()TD$FIG]
L.H. Shen et al. / Chinese Chemical Letters 22 (2011) 768–770
769
CH3O
CH3O
CH3O
NHCOCH3
O
NHCOCH3
CH3O
OCH3
OCH3
O
OCH3
1
NHCH3
2
CH3O
CH3O
CH3O
CH3O
NHCO(CH2)2CO2R1Br
c
CH3O
CH3O
NH2
O
NHCO(CH2)2CO2H
O
b
a
OCH3
2
OCH3
O
OCH3
NHCH3
NHCH3
5a-5f
3
NHCH3
4
d
f
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
NHCO(CH2)2CO2
O
Br
NHCO(CH2)2CO2R1ONO2
O
NHCO(CH2)2CO2
O
e
R2
OCH3
OCH3
6a-6f
OCH3
NHCH3
NHCH3
NHCH3
8a-8b
R1= (CH2)2, (CH2)3, (CH2)4 , (CH2)5
(CH2)6, CH2CH=CHCH2
8a R2=para-CH2ONO2
8b R2=meta-CH2ONO2
7a-7b
Scheme 1. Reagents and conditions: a, conc. H2SO4, H2O, reflux, 5 h; b, succinic anhydride, pyridine, 60 8C, 5 h, 95%; c, BrR1Br, K2CO3, DMF, rt,
5 h, 80–85%; d, AgNO3, CH3CN, reflux, 15 h, 88–95%; e, (i) SOCl2, DMF (cat), reflux, 5 h; (ii) p- or m-hydroxybenzyl bromide, CH2Cl2, rt, 12 h,
71% and 78%; f, AgNO3, CH3CN, reflux, 15 h, 45% and 50%.
In 2009, Chang et al. reported a group of nitrate derivatives of Col, which have proved that the structure
modification of the 10 positions does not interfere with the molecular recognition of Col [12]. Succinic acid is a low
toxicity agent and can help in the compounds forming prodrug effectively by ester bond [13,14]. With these in mind,
and also based on the diverse bioactivities of the above-mentioned Col, we recently conjugated N-methyl
colchiceinamide with nitrate using succinic acid as a linker to synthesize a series of novel NO-releasing derivatives of
Col, hoping that these derivatives might be transported to target site where they would release active compounds and
high concentrations of NO to selectively kill tumor cells without affecting normal cells.
The synthetic route of these target compounds is outlined in Scheme 1. Col 1 was purchased from Nanjing Tianzun
Chemicals Co., Ltd., China, with over 98% purity. The lead compound 2 was prepared from 1, according to the
literature [15] in 90% yield.
N-methyl colchiceinamide 2 was refluxed with concentrated H2SO4 in water to give N-methyl deacetylcolchi-
ceinamide 3. Compound 3 was acylated by succinic anhydride in dry pyridine at 60 8C to give succinate 4 in 95%
yield. 4 was treated with dibromoalkanes in the presence of K2CO3 and DMF at room temperature to generate
important intermediates 5a–5f in 80–85% yields. Compounds 5a–5f were further converted to the corresponding
target compounds 6a–6f with AgNO3 in CH3CN in good yields (88–95%).
In a similar way, compounds 7a and 7b were prepared in good yields from 4 via chlorination by SOCl2 to form acid
chloride, followed by condensation with p-hydroxybenzyl bromide and m-hydroxybenzyl bromide respectively in the
presence of CH2Cl2 at room temperature, subsequent reaction with AgNO3 in CH3CN gave target compounds 8a and 8b.
The resulting products were purified by column chromatography and their structures are shown in Scheme 1 and the
data of yield, MS, IR and 1H NMR spectra and elemental analysis (EA) of selected compounds are shown in Ref. [16].
The cytotoxic activity of all target compounds in vitro was determined by MTT assay [17], using Col as a positive
control, and the result is summarized in Table 1. Four different cell lines were used: A2780 (human ovary cancer),
A549 (human lung cancer), BEL7402 (human hepatoma), MCF7 (Human breast carcinoma).
The study results indicate that these novel nitrate derivatives showed superior or comparable cytotoxic activity to
Col in vitro. For human ovary cancer cell line (A2780) and human lung cancer cell line (A549), compounds 6c, 8a and
8b have better cytotoxicity than Col, and compounds 6b and 6d have similar cytotoxicity as Col. In human hepatoma
cell line (BEL7402), compounds 6b, 6d and 6e have similar cytotoxicity as Col, whereas compounds 6c, 8a and 8b
have more potent cytotoxicity than Col. As to human breast carcinoma cell line (MCF7), compound 6c exhibited
almost tenfold potent activities than Col. The results demonstrated that NO-donating Col derivatives could really
improve colchicine’s antitumor activity in vitro.

770
L.H. Shen et al. / Chinese Chemical Letters 22 (2011) 768–770
Table 1
The cytotoxicity data of the target compounds.
Compd.
IC50 (mmol/L)/cell line
A2780
A549
BEL7402
MCF7
6a
6b
6c
6d
6e
6f
0.104
0.099
0.010
0.096
0.109
0.205
0.027
0.036
0.094
0. 106
0.088
0.012
0.086
0.205
0.135
0.038
0.049
0.078
0.202
0.085
0.013
0.085
0.086
0.120
0.029
0.037
0.080
0.103
0.135
0.008
0.100
0.095
0.202
0.036
0.058
0.084
8a
8b
Col
In summary, a series of Col–nitrate conjugates were synthesized and evaluated for their in vitro cytotoxicity against
four human tumor cell lines. It was found that 6c showed significant cytotoxic activities. Further biological evaluations
are currently in progress and will be reported in due course.
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[16] The data of selected compounds: 6a: yield 90.0%, IR (KBr, n): 3444, 2907, 1733, 1630, 1490, 1455, 1414 cmÀ1; 1H NMR (CDCl3, 300 MHz):
d 1.50–3.40 (m, 9H), 3.07 (d, 3H, N–CH3), 3.67 (s, 3H, MeO-1), 3.91 (s, 3H, MeO-2), 3.95 (s, 3H, MeO-3), 4.25–4.29 (t, 2H, J = 6.0 Hz,
OCH2), 4.44–4.48 (t, 2H, J = 6.0 Hz, OCH2), 4.60–4.72 (m, 1H, H-7), 6.54 (s, 1H, H-4), 7.34 (m, 2H, Ar-H), 7.49 (s, 1H), 8.71 (bs, 1H, NHCO);
MS (ESI, m/z): 545.3 [M]+; Anal. Calcd. for C26H31O10N3: C 57.24, H 5.73, N 7.70; Found: C 57.28, H 5.78, N 7.63. 6c: yield 92.0%, IR (KBr,
n): 3450, 2925, 1738, 1630, 1598, 1506, 1447 cmÀ1; 1H NMR (CDCl3, 300 MHz):d 1.50–3.38 (m, 13H), 3.07 (d, 3H, N–CH3), 3.66 (s, 3H,
MeO-1), 3.90 (s, 3H, MeO-2), 3.94 (s, 3H, MeO-3), 4.24–4.28 (t, 2H, J = 6.0 Hz, OCH2), 4.44–4.48 (t, 2H, J = 6.0 Hz, OCH2), 4.65–4.72 (m,
1H, H-7), 6.52 (s, 1H, H-4), 7.27 (m, 2H, Ar-H), 7.49 (s, 1H), 8.93 (bs, 1H, NHCO); MS (ESI, m/z): 573.4 [M]+; Anal. Calcd. for C28H35O10N3:
C 58.63, H 6.15, N 7.33; Found: C 58.65, H 6.26, N 7.39. 8a: yield 45%, IR (KBr, n): 3445, 2920, 1739, 1630, 1597, 1510, 1455, 1322 cmÀ1; 1H
NMR (CDCl3, 300 MHz): d 1.50–3.37 (m, 9H), 3.07 (d, 3H, N–CH3), 3.65 (s, 3H, MeO-1), 3.89 (s, 3H, MeO-2), 3.93 (s, 3H, MeO-3), 4.68 (m,
1H, H-7), 5.47 (s, 2H, OCH2), 6.52 (s, 1H, H-4), 6.93 (m, 4H, Ar-H), 7.30 (m, 2H, Ar-H), 7.49 (s, 1H), 9.07 (bs, 1H, NHCO); MS (ESI, m/z):
607.2 [M]+; Anal. Calcd. for C31H33O10N3: C 61.28, H 5.47, N 6.92; Found: C 61.35, H 5.52, N 6.89.
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