Synthesis of novel spin-labelled combretastatin A-4 derivatives as potential antineoplastic agents

Article abstract of DOI:10.1080/14786419.2011.566222

Four novel spin-labelled combretastatin A-4 (CA-4) analogues were first synthesised in quantitative yield by reacting CA-4 with the corresponding nitroxyl radical. Their cytotoxic activities against A-549 (human lung cancer) and HePG-2 (human liver cancer) invitro were evaluated, and the results indicated that these derivatives were more cytotoxic than the clinical drug irinotecan.

Full text of DOI:10.1080/14786419.2011.566222

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Synthesis of novel spin-labelled
combretastatin A-4 derivatives as
potential antineoplastic agents
Xiao-Jing Li a , Liu Yang b , Ying-Qian Liu a & Chong Li a
a School of Pharmacy, Lanzhou University, Lanzhou 730000, P.R.
China
b Environmental and Municipal Engineering School, Lanzhou
Jiaotong University, Lanzhou 730000, P.R. China
Published online: 19 Oct 2011.
To cite this article: Xiao-Jing Li , Liu Yang , Ying-Qian Liu & Chong Li (2012) Synthesis of novel
spin-labelled combretastatin A-4 derivatives as potential antineoplastic agents, Natural Product
Research: Formerly Natural Product Letters, 26:14, 1271-1277, DOI: 10.1080/14786419.2011.566222
To link to this article: http://dx.doi.org/10.1080/14786419.2011.566222
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Natural Product Research
Vol. 26, No. 14, July 2012, 1271–1277
Synthesis of novel spin-labelled combretastatin A-4 derivatives
as potential antineoplastic agents
a
Xiao-Jing Li , Liu Yang , Ying-Qian Liu and Chong Li
b
a
a
*
aSchool of Pharmacy, Lanzhou University, Lanzhou 730000, P.R. China;
bEnvironmental and Municipal Engineering School, Lanzhou Jiaotong
University, Lanzhou 730000, P.R. China
(Received 29 September 2010; final version received 23 February 2011)
Four novel spin-labelled combretastatin A-4 (CA-4) analogues were first
synthesised in quantitative yield by reacting CA-4 with the corresponding
nitroxyl radical. Their cytotoxic activities against A-549 (human lung
cancer) and HePG-2 (human liver cancer) in vitro were evaluated, and the
results indicated that these derivatives were more cytotoxic than the clinical
drug irinotecan.
Keywords: spin-label; nitroxyl radical; combretastatin A-4; antineoplastic
agents
1. Introduction
The nitroxyl free radicals have a wide range of activities in biology. Previous studies
have shown that the introduction of nitroxyl moiety can lead to a series of positive
effects, such as higher alkylating ability, lower carbamoylating activity, better
antimelanomic activity and lower general toxicity (Ankel, Lai, Hopwood, &
Zivkovic, 1987; Jin, Chen, & Tian, 2006; Sosnovsky & Li, 1985). These types of
compounds can move through cell membranes as a transport (Li, Zhang, Wu, &
Tian, 2002; Zheleva, & Gadjeva, 2001). In addition, the nitroxyl free radicals possess
low toxicity and are not mutagenic or carcinogenic by themselves (Gadjeva, 2002;
Sosnovsky, 1990; Tian, Zhang, & Li, 2002). In our previous studies, we have
synthesised a series of spin-labelled podophyllotoxin derivatives and found that
introduction of a stable nitroxyl radical into podophyllotoxin or its analogues could
increase antitumour activity and cause marked decrease in toxicity compared with
parent compounds (Liu & Tian, 2005; Liu, Yang, & Tian, 2007; Yang, Liu, Tan, Li,
& Tian, 2007). The same conclusion also can be found in the spin-labelled
camptothecin derivatives synthesised by our lab (Liu, Tian, Yang, & Zhan, 2008;
Liu, Yang, Zhao, & Li, 2010).
Combretastatins are mitotic agents isolated from the bark of the South African
tree Combretum caffrum (Hamel & Lin, 1983; Pettit, Cragg, Herald, Schmidt, &
Lobavanijaya, 1982; Pettit et al., 1989). The most potent of these is combretastatin
*Corresponding author. Email: yqliu@lzu.edu.cn
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2011.566222
http://www.tandfonline.com

1272
X.-J. Li et al.
COOH
H3CO
H3CO
COOH
CHO
H3CO
H3CO
a
b
+
OCH3
OH
OH
OCH3
H3CO
OCH3
OCH3
OCH3
OH
OCH3
OCH3
6
3
4
5
Reagents and conditions: (a) AC2O/Et3
Scheme 1. Synthesis of CA-4 (1).
A-4(CA-4, 1), a natural stilbenoid, which has been found to be a new vascular
targeting agent molecule known for its antitumour activity due to its anti-tubulin
properties (Boye & Brossi, 1992; Griggs, Metcalfe, & Hesketh, 2001; Lin, Ho, &
Pettit, 1989; Lin et al., 1988). Its water-soluble phosphate prodrug (CA-4P, 2) is now
in phase II of clinical trials (Pettit & Rhodes, 1998; Pettit et al., 1995).
Combretastatin A-4 is thus attractive as a lead compound for development of
anticancer drugs.
Based upon the above results, as part of our programme aimed at the discovery
and development of bioactive molecules derived from natural products, in this study,
four novel spin-labelled combretastatin A-4 derivatives (8a–d) were designed,
synthesised and preliminarily evaluated for their cytotoxic activities in vitro against
A-549 (human lung cancer) and HePG-2 (human liver cancer).
2. Results and discussion
2.1. Syntheses
The synthesis of CA-4 is shown in Scheme 1. Base-catalysed condensation of 3,4,5-
trimethoxyphenylacetic acid (two equivalents, 3) with 3-hydroxy-4-methoxybenzal-
dehyde (one equivalent, 4), in the presence of triethylamine and acetic anhydride at
140ꢀC, gave the carboxylic acid 5. Decarboxylation of the acid 5 was achieved using
powdered copper and quinoline at 230ꢀC, following the method of Molho and
Coillard (1956). Initially, cis-CA-4 (1) was purified using flash column chromatog-
raphy. A small amount of the trans-isomer was also isolated, but on repetition of the
reaction, it was found that the cis-isomer could be purified by recrystallisation alone.
Clearly, this represents an efficient stereoselective synthesis of cis-CA-4, which
avoids the use of protecting groups and chromatography.
The spin-labelled combretastatin A-4 has been achieved in quantitative yield by a
simple modification of N,N0-dicyclohexylcarbodiimide (DCC) and 4-dimethylami-
nopyridine (DMAP) to give the desired compounds 8a–d (Scheme 2) under a
nitrogen atmosphere at 0ꢀC. Synthesised target compounds were characterised by
melting point, IR and HRMS spectral analyses. In order to obtain precise three-
dimensional structural information and absolute configuration of CA-4, it was
grown from a mixture of ethyl acetate and hexane and its single crystal structure
determined by X-ray crystallography, as illustrated in Figure 1.

Natural Product Research
1273
H3CO
H3CO
H3CO
O
DCC, DMAP
CH2Cl2
H3CO
O
+
OCH3
OCH3
R
OH
OH
OCH3
O
R
OCH3
8a–8d
1
7a–7d
R=
N
N
N
N
O.
O.
O.
O.
a
c
b
d
Scheme 2. Synthesis of compounds 8a–d.
MeO
MeO
MeO
MeO
O
P
OMe
OMe
OH
O
ONa
ONa
OMe
OMe
Combretastatin A-4(1)
CA-4P(2)
X-ray crystal structure of 1
Figure 1. Chemical structures of compounds 1 and 2, and X-ray crystal structure of 1.
2.2. Biological activity
The biological activities of compounds 8a–d were evaluated by an in vitro
cytotoxicity test, which was carried out with a panel of two tumour cell lines that
comprise human lung cancer (A-549) and human liver cancer (HePG-2). The results
are summarised in Table 1.
As illustrated in Table 1, in the two tumour cell lines, compounds 8a–d exhibited
higher inhibitory activities compared to irinotecan. Also, from screening, it was
observed that all the tested compounds showed selective activities against human
lung cancer A-549. For example, the IC50 value of compound 8c for A-549 is
0.15 mM, while that of compound 8c for HePG-2 is 3.58 mM. However, different
nitroxyl radicals in this class of compounds did not show obviously different effects
on the inhibition of the two tumour cell lines in vitro. As it can be seen, these
analogues have exhibited an interesting profile of in vitro cytotoxic activity. The IC50
values of these analogues were either similar or better than those of the parent
compound 1. Meanwhile, compounds 8a–d as prodrugs would likely be in vivo
activated to release the toxic parent CA-4 and such an assumption was supported by
our previous research (Liu et al., 2008).The results indicated that the design and
synthesis of these compounds should be beneficial for obtaining the therapeutic
values of CA-4 and they probably have synergistic action to tumour cell lines.

1274
X.-J. Li et al.
Table 1. In vitro cytotoxic activities of compounds 8a–d.
IC50 (mM)a
Compounds
A-549b
HePG-2b
8a
8b
8c
8d
0.16
0.042
0.15
0.17
0.035
2.00
3.27
1.83
3.58
11.22
2.39
34.07
1
Irinotecan
a
Note: The value is the average of triplicate.
bMTT method, drug exposure was for 48 h.
3. Experimental
Melting points were taken on a Kofler melting point apparatus and are uncorrected;
IR spectra were obtained on NIC-NEXUS 670 spectrophotometer; mass spectral
analysis was performed on a Bruker Daltonics APEXII49e instrument with ESI
source as ionisation. The stable nitroxide free radicals were prepared by following a
modified previous procedure (Krinitskaya, 1980; Wong, Chwenk, & Hsia, 1974).
3.1. Synthesis of cis-combretastatin A-4 (1)
A mixture of 3,4,5-trimethoxyphenylacetic acid 3 (2 g, 8.84 mmol) and 3-hydroxy-4-
methoxybenzaldehyde 4 (0.67 g, 4.4 mmol), acetic anhydride (4 mL) and triethyla-
mine (2 mL) were heated under reflux for 3 h. After acidification with concentrated
hydrochloric acid (6 mL), the resulting solid was filtered off and recrystallised from
ethanol to give acrylic acid intermediates 5 (950 mg, 60%) as fine yellow needles.
Subsequently, the corresponding acrylic acid intermediates 5 (2 g, 5.56 mmol) were
added to powdered copper (28.8 mmol) in quinoline (20 mL) and the resulting
mixture heated at 200ꢀC for 2 h. Upon cooling, ether was added and the copper
filtered off through Celite. The filtrate was washed with 1 M hydrochloric acid, and
the aqueous layer separated and extracted with ether. The combined organic layers
were washed with saturated aqueous sodium carbonate, water, brine, dried (MgSO4)
and concentrated in vacuo. Flash column chromatography (SiO2 petroleum: ethyl
acetate 7:3) and recrystallisation from ethyl acetate and petrol gave 1 as a pale yellow
crystalline solid (1.19 g, 68%).
Yield: 65%; m.p. 117–118ꢀC. IR (KBr) (cmꢁ1): 3453, 3006, 1616, 1580, 1327,
1238, 1126 and 1010; 1H-NMR (400 MHz, CDCl3) ꢀ: 3.69 (s, 6H), 3.84 (s, 3H), 3.89
(s, 3H), 5.53 (s, 1H), 6.42 (d,1H, J ¼ 12.4 Hz), 6.47 (d,1H, J ¼ 12.4 Hz), 6.55 (s, 2H),
6.75 (d, 1H, J ¼ 8.3), 6.82 (dd,1H, J ¼ 8.3, 1.9 Hz) and 6.92 (1H, d, J ¼ 1.9 Hz); 13C-
NMR (400 MHz, CDCl3) ꢀ: 153.07, 146.02, 145.45, 137.34, 132.93, 130.81, 129.71,
129.22, 121.32, 115.27, 110.56, 106.28, 61.15 and 56.15; MS m/z (%): 316 (Mþ, 100),
301 (75), 241 (8), 226 (6), 211 (5), 142 (12), 115 (8), 93 (5) and 57 (8); Anal. Calcd for
C18H20O5: C, 68.40% and H, 6.38%. Found: C 68.3.7% and H 6.30%.

Natural Product Research
1275
3.2. Synthesis of general procedure for synthesis of target compounds (8a–d)
A stirred solution of 1 (200 mg, 0.633 mmol), nitroxide (0.633 mmol) and DMAP
(8 mg, 61 mmol) in anhydrous dichloromethane (15 mL) under an atmosphere of
nitrogen in the dark was cooled to 0ꢀC, DCC (157 mg, 0.76 mmol) was added and the
reaction mixture stirred at room temperature for 48 h. Then, the mixture was filtered,
evaporated and the residue purified by column chromatography with petroleum/
ethyl acetate as eluent. Synthesised target compounds 8a–8d were characterised by
melting point, IR, MS and HRMS spectral analyses.
3.2.1. 3,4,5,40-tetramethoxy-30-O-(10-oxyl-30-carbonyl-20,20,50,50-tetramethylpyrro-
line)-Z-stilbene (8a)
Yellow powder, yield: 65%; m.p. 112–114ꢀC. IR (KBr): 2922, 2846, 1735, 1613, 1578,
1511, 1460, 1365, 1324, 1239, 1126 and 1021; MS m/z 483 (M þ Hþ); HRMS: m/z
Calcd for C27H33NO7: 483.2257 [M þ H]þ, Found: 483.2264 [M þ H]þ.
3.2.2. 3,4,5,40-tetramethoxy-30-O-(10-oxyl-30-carbonyl-20,20,50,50-tetramethyl pyrro-
lidine)-Z-stilbene (8b)
Yellow powder, yield: 72%; m.p. 102–104ꢀC. IR (KBr) 2925, 2850, 1711(C¼O),
1613, 1579, 1509, 1460(ArH), 1363(N–O), 1327, 1241, 1129 and 1020; MS m/z 485
(M þ Hþ); HRMS: m/z Calcd for C27H35NO7: 485.2414 [M þ H]þ, Found: 485.2413
[M þ H]þ.
3.2.3. 3,4,5,40-tetramethoxy-30-O-(10-oxyl-40-carbonyl-20,20,60,60-tetramethylpiper
dinylidene)-Z-stilbene (8c)
Red powder, yield: 67%; m.p. 109–111ꢀC. IR (KBr) 2932, 2851, 1712(C¼O), 1613,
1579, 1509, 1461(ArH), 1360(N–O), 1329, 1241, 1127 and 1026; MS m/z 497
(M þ Hþ); HRMS: m/z Calcd for C28H35NO7: 497.2414 [M þ H]þ, Found: 497.2432
[M þ H]þ.
3.2.4. 3,4,5,40-tetramethoxy-30-O-(10-oxyl-40-carbonyl-20,20,60,60-tetramethylpiperi-
dine)-Z-stilbene (8d)
Red powder, yield: 60%; m.p. 93–95ꢀC. IR (KBr) 2925, 2852, 1713(C¼O), 1612,
1580, 1508, 1460(ArH), 1362(N–O), 1326, 1223, 1129 and 1025; MS m/z 498 (Mþ);
HRMS: m/z Calcd for C28H37NO7: 499.2570 [MþH]þ, Found: 499.2574 [M þ H]þ.
3.3. Cytotoxicity assays
Cells were incubated at 37ꢀC in a 5% CO2 atmosphere. The 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to deter-
mine growth inhibition (Mosmann, 1983). The spin-labelled derivatives were
dissolved in saline for five concentrations (0.005–50 mM). For the A-549 and
HePG-2, cells were plated in 96-well plates, allowed to attach for 24 h and then
exposed in quadplex well for 48 h. The media were aspirated, and 10 mL of
5 mg mLꢁ1 MTT solution (dilute in sterile phosphate-buffered saline) diluted in

1276
X.-J. Li et al.
serum-free media was added to each well. After 4 h of incubation, the solution was
centrifuged for 10 min under 2000 rpm. The supernatant was mixed with 150 mL
DMSO, and then shaken on an oscillator. The absorbance at l570 was determined on
a plate reader. IC50 values were determined from a log plot of percent of control
versus concentration.
4. Conclusions
In summary, novel spin-labelling of CA-4 derivative is a promising direction in
antitumour chemotherapy, not only because they exhibit superior activities, but also
because they can be monitored by electron spin resonance in pharmacological
experiments. In this study, four spin-labelled combretastatin A-4 derivatives were
first synthesised, and they exhibited significant cytotoxic activities against two
tumour cell lines. Further biological evaluation is in progress to define their tubulin
polymerisation activity and to clarify whether spin-labelled combretastatin A-4
analogues might counteract the toxicity of this class antitumour drug.
Acknowledgements
This study was financially supported by the National Natural Science Foundation of China
(30800720); the Fundamental Research Funds for the Central Universities (860120); and the
Post-Doctor Research Foundation (20090450142).
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