6428 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Brief Articles
carbonate (2 × 5 mL), and brine (5 mL) and dried over sodium
sulfate. After concentration under reduced pressure, the residue was
purified by silica gel column chromatography (petroleum ether-
EtOAc, 6:4) to furnish the corresponding final compounds 5a-t.
inversely with the size of the halogen. The inactivity of the
p-diphenyl derivative 5t indicates that a bulky substituent on
the same position is detrimental for activity, a conclusion
supported by the finding with the halides by a comparison of
the reduced activity of a p-ethoxy substituent relative to the
p-methoxy analogue and by the effect of a p-NO2 substituent.
There was also no clear difference in effect on activity between
an EWG versus an ERG, once substituent size was taken into
account. We identified tubulin as the molecular target of the
compounds, because those with the greatest inhibitory effects
on cell growth strongly inhibited tubulin assembly and binding
of colchicine to tubulin. These activities differed little from those
of CA-4, and all active compounds had quantitatively similar
effects in the tubulin assays, varying within a narrow range
(IC50s for assembly, 0.64 to 1.5 µM with 10 µM tubulin; 53-
81% inhibition of the binding of 5 µM colchicine, with the
inhibitor and tubulin both at 1 µM). We also showed by flow
cytometry that four of the active compounds had cellular effects
typical of agents that bind to tubulin, causing accumulation of
cells in the G2/M phase of the cell cycle and a substantial
increase in the number of apoptotic cells. These compounds
constitute a new class of potent antitubulin agents with the
potential to be developed clinically for anticancer chemotherapy.
Supporting Information Available: Detailed biological pro-
tocols, physical and spectroscopic data for compounds 5a-t and
7a-t, and elemental analyses. This material is available free of
References
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Experimental Section
General Procedure A for the Synthesis of Compounds 7a-t.
Neat POCl3 (12.27 g, 80 mmol) was added dropwise over 30 min,
maintaining the temperature below 25 °C, to a stirred solution
cooled on ice, of the appropriate acetophenone derivative (20 mmol)
dissolved in DMF (60 mL). The reaction mixture was stirred for 2
h at room temperature, and NH2OH‚HCl (1.4 g, 20 mmol) was
added portionwise, resulting in an exothermic reaction. The mixture
was stirred overnight, cold water (100 mL) was added, and the
aqueous phase was extracted with ethyl acetate (3 × 50 mL). The
organic phase was washed with brine (50 mL), dried over Na2SO4,
and concentrated in vacuo. The resulting precipitate was purified
by silica gel column chromatography to yield 3-chlorocinnamoni-
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Synthesis of O-Ethyl Ester of S-[2-Oxo-2-(3,4,5-trimethox-
yphenyl)-ethyl] Dithiocarbonic Acid (8). A solution of 2-bromo-
1-(3,4,5-trimethoxyphenyl)-ethanone (5.78 g, 20 mmol) in 20 mL
of acetone was added dropwise over 30 min to a solution of
potassium ethyl xanthate (4 g, 25 mmol) dissolved in 50 mL of
acetone. The reaction mixture was stirred for 1 h at room
temperature. Acetone was removed in vacuo, and the residue was
suspended in dichloromethane (40 mL). The resulting mixture was
washed with water (3 × 10 mL) and brine (10 mL), dried over
sodium sulfate, and concentrated in vacuo. The resulting precipitate
was purified by column chromatography eluting with petroleum
ether-ethyl acetate (8:2, v/v). Compound 8 was isolated as a white
1
solid. Yield 74%; mp 58-60 °C. H NMR (CDCl3): δ 1.41 (t, J
) 7.2 Hz, 3H), 3.93 (s, 9H), 4.65 (m, 4H), 7.28 (s, 2H).
General Procedure B for the Synthesis of 5-Aryl/heteroaryl-
3-amino-thiophen-2-yl-(3,4,5-trimethoxyphenyl)-methanones 5a-
t. Piperidine (0.31 mL, 3 mmol) was added to a stirred solution of
dithiocarbonic acid O-ethyl ester S-[2-oxo-2-(3,4,5-trimethoxyphe-
nyl)-ethyl] ester 8 (1.5 mmol) dissolved in ethanol (30 mL). The
reaction mixture was stirred for 30 min at room temperature. The
corresponding 3-chloroacrylonitrile (1.5 mmol) was added, and the
solution was stirred at reflux for 3 h, after which ethanol was
removed under reduced pressure and the residue dissolved in
dichloromethane (20 mL). The organic phase was washed with a
5% v/v solution of HCl (2 × 5 mL), a saturated solution of sodium
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B. W.; Hamel, E. Structure-activity analysis of the interaction of
curacin A, the potent colchicine site antimitotic agent, with tubulin
and effects of analogues on the growth of MCF-7 breast cancer cells.
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JM060804A
Romagnoli, Romeo
