Synthesis and Antitumor Activity of Combretastatin Analogues

 

Sunil Kumar¹, Surrinder Koul², Ajay Kumar Meena⁴, A.K. Saxena³, O.P. Suri¹ and K.L. Dhar¹*

¹Department of Pharmaceutical Chemistry, I. S. F. College of Pharmacy, Moga, 142 001, India,

²Department of Bio-organic Chemistry, IIIM Jammu Tawi 180001, India

³Division of Pharmacology, IIIM Jammu Tawi, 180001 India

⁴National Institute of Ayurvedic Pharmaceutical Research, Moti Bagh Road, Patiala, India.

*Corresponding Author E-mail: dharkl@yahoo.com

ABSTRACT:

Plants, source of various bioactive compounds have played crucial role in development of several clinically useful anticancer agents’ viz., vincristin, vinblastin, camptothecin derivatives and taxol. Combretastatin, a potent anticancer molecule was first isolated from plant. In this continuation a series of combretastatin analogues has been synthesized on the basis of our butterfly model with two wings as aryl groups and connecting carbon chain as the body. In present study we have synthesized combretastatin analogues of different substitutions, showed potent anticancer activity against different cell lines in different fashion according to substitution. Compound 6 showed marked anticancer activity against colo-205 cell lines (colon cancer).

 

 

 

INTRODUCTION:

In 1982, Petit and co-workers isolated oxygenated stilbene derivatives from the bark of African willow tree Combretum caffrum, compound CA4 (Fig.1) appears to be the most powerful antimitotic agent in the series and is remarkably simple in the chemical structure and the most potent tubulin inhibitor⁽¹⁾. The newly isolated compounds were named combretastatins and identified as natural products with remarkable biological properties. Combretastatin bind to distinct sites in the β-tubulin subunit by interfering with the microtubule functions. In vitro, these agents arrest cell division in mitosis, eventually leading to cell death⁽²⁾. Syntesis and biological activity of numerous analogues with a five membered heterocycle as as linker of two aromatic of combretastain has been reported⁽³⁾. Our group has developed combretastatin, an important class of antimitotic agents⁽⁴⁾. Comprehensive structure activity relationship study of combretastatin has been reported to inhibit the tubulin assembly by binding to colchicines binding site. The antimitotic agent, combretastatin, binds to β-tubulin and inhibits tubulin assembly. Since this observation in the early 1970s, the mechanistic details of this interaction have often been addressed⁽⁵⁾.

 

Fig.1

 

RESULTS AND DISCUSSION:

Numbers of stilbene derivatives (Fig.2) were prepared by condensation of phenyl acetic acid with different substituted aldehydes (Fig.3). The compounds were purified by column chromatography. The yields of synthesized compounds ranged between 49-60%. The structures of the synthesized compounds were confirmed by spectral data (IR, NMR and MS). All compounds were evaluated for their pharmacological activity (Table-1) and compound 6 showed significant activities in colon cancer (colo-205 cell line).

 

Structure of compounds:

Fig.2

 

 

1, R₂, R₄, R₅=OCH₃; R_3, R₆=H

2, R₂=NO₂; R₃, R₄, R₅, R₆=H

 

 

4, R₂, R₅=OCH₃; R₃, R₄, R₆=H

5, R₂, R₃, R₄=OCH₃; R₅, R₆=H

 

EXPERIMENTAL:

Fig. 3

 

A. Acetic anhydride,

B. Triethyl amine

 

General procedure for the preparation of acrylic acids (1 to 6):

A mixture of phenyl acetic acid (1g, 7.36 mmol), substituted benzaldehyde (7.36 mmol) and triethylamine (2ml) in Ac2O (4 ml) was heated 6 to 8 hours. After cooling, the reaction mixture was acidified with 35% aqueous HCl (6ml) and kept at room temperature overnight. The precipitate was collected by filtration and purified by column chromatography using silica gel 60-120 and 30% ethyl acetate in hexane as mobile phase.

 

Spectral data:

(E)-3-(2,4,5-trimethoxyphenyl)-2-phenyl-prop-2-en oic acid (1):

Yield: 49. 0%; m. p. 182-184; IR ν_max cm^-1 (KBr, ): 3477, 1668, 1603, 1506, 1264; ¹HNMR (CDCl₃) δ: 3. 18(3H, s, Ar-OCH₃), 3. 87(6H, s, Ar-OCH₃), 6. 26(H, s, Ar-H), 6. 43(H, s, Ar-H) 7. 3-7. 4(5H, m, 5xAr’-H), 8. 3(1H, s, =CH); ¹³C NMR (CDCl₃): 52. 3, 53. 0, 55. 6, 121. 1, 126. 6, 128. 0, 128. 2, 128. 9, 129. 8, 142. 2, 151. 7, 152. 6, 156. 9, 159. 7, 164. 5, 174. 8. MS/MS: 337 (M+Na)⁺

 

(E)-3-(2-nitrophenyl)-2-phenyl-prop-2-en oic acid (2):

Yield: 58.0%; m.p. 182-184; IR ν_max cm^-1 (KBr,): 3411, 1670, 1617, 1579, 1506, 1421; ¹HNMR (CDCl₃) δ: 6.3-6.9 (4H, m, Ar-H), 7.6-7.2(5H, m, 5xAr’-H), 8.16 (1H,s,=CH); ¹³C NMR (CDCl₃): 123.3, 124.8, 126.6, 128.0, 128.3, 128.8, 129.1, 130.2, 130.5, 133.5, 140.4, 167.9 MS/MS: 292(M+Na)⁺

 

(2Z,4E)-2,5-diphenylpenta-2,4-dien oic acid (3):

Yield: 58.0%; m.p. 182-184; IR ν_max cm^-1 (KBr,): 3446, 1678, 1621, 1579, 1346; ¹HNMR (CDCl₃) δ: 5.59 (1H, d, j=15.2, =CH₂), 5.96 (1H, dd, j=15.2 & j=14.8, =CH₂), 6.8 (1H, d, j=15.2, =CH₂), 7.4-7.2(5H, m, 5xAr-H), 7.7-7.5(5H, m, 5xAr’-H) 7.8(1H,s,=CH); ¹³C NMR (CDCl₃): 120.2, 122.1, 122.8, 123.0, 123.3, 124.2, 126.6, 128.0, 128.2, 128.9, 129.8, 135.5, 135.9, 144.2, 164.5. MS/MS: 273 (M+Na)⁺

 

(E)-3-(2,5-dimethoxyphenyl)-2-phenyl-prop-2-en oic acid (4):

Yield: 59.7%; m.p. 147-149; IR ν_max cm^-1 (KBr,): 3437, 1666, 1580, 1260; ¹HNMR (CDCl₃) δ: 3.2(3H, s, OCH₃), 3.9(3H, s, -OCH₃), 6.2(1H, d, j=7.9 Hz, Ar-H), 6.75(1H, d, j=7.9 Hz, Ar-H) 7.3-7.2(5H, m, 5XAr’-H), 8.3(1H, s, =CH);  ¹³C NMR (CDCl₃): 50.6, 54.3, 119.0, 121.0, 125.0, 127.5, 129.6, 130.0, 132.0, 133.8, 135.2, 138, 139.7, 139.0, 145.1, 146.7, 167.6. MS/MS: 307 (M+Na)⁺

 

(E)-3-(2,3,4-trimethoxyphenyl)-2-pheny-prop-2en oic acid (5):

Yield: 58.0%; m.p. 182-184; IR ν_max cm^-1 (KBr,): 3411, 1667, 1491, 1258; ¹HNMR (CDCl₃) δ: 3.7(3H, s, Ar-OCH₃), 3.8(3H, s, Ar-OCH₃), 3.9(3H, s, Ar-OCH₃), 6.3 (1H, d, j=8.8, Ar-H), 6.4 (1H, d, j=8.8, Ar-H), 7.4-7.2(5H, m, 5xAr’-H), 8.2 (1H,s,=CH); ¹³C NMR (CDCl₃): 52.9, 53.8, 55.6, 121.1, 126.6,128.0, 128.2, 128.9, 129.8, 140.2, 151.7, 152.6, 156.9, 159.7, 164.5, 174.5. MS/MS: 337 (M+Na)⁺

 

(E)-3-(naphthalene-5yl)-2-phenyl-prop-2-en oic acid (6):

Yield: 53.0%; m.p. 182-184; IR ν_max cm^-1 (KBr,): 1677, 1614, 1579, 1506, 1260; ¹HNMR (CDCl₃) δ: 7.4-7.2 (5H, m, Ar-H), 8.4-8.1 (7H, m, 5xAr’-H), 8.5 (1H,s,=CH); ¹³C NMR (CDCl₃): 120, 123, 124.9, 125.3, 127.3, 127.9, 128.6, 129.3, 130, 130.4, 131.2, 131.5, 131.8, 133.5, 142.2, 146.3, 169.1. MS/MS: 297 (M+Na)⁺

 

Anticancer activity: In vitro cytotoxicity against five human cancer cell lines was determined using 96-well tissue culture plate⁽⁶⁾. The cells were allowed to grow in carbon dioxide incubator (37ºC) for 24 hours. Test materials in complete growth medium (100µl) were added after 24 hours of incubation to the wells containing cell suspension. The plates were further incubated for 48 hours in a carbon dioxide incubator. The cell growth was stopped by gently layering trichloroacetic acid (50%, 50µl) on top of the medium in all the wells. The plates were incubated at 4^oC for one hour to fix the cells attached to the bottom of the wells. The liquid of all the wells was gently pipetted out and discarded.

Table-1 Anticancer activity data of compounds 1 – 6

Cell line types			PC-3	Colo-205		THP-1		A-549
Tissue			Prostrate	colon		Leukemia		Lung
CCL  Code	Compound Code	Conc. in mole	%GROWTH INHIBITION
M-4092	1	5×10-5	4		5	0	0
		1×10-4	28		16	21	20
M-4093	2	5×10-5	10		32	0	15
		1×10-4	22		60	0	25
M-4094	3	5×10-5	0		15	8	10
		1×10-4	9		44	35	43
M-4095	4	5×10-5	0		0	0	0
		1×10-4	0		0	4	10
M-4096	5	5×10-5	0		0	0	0
		1×10-4	7		0	0	0
M-4097	6	5×10-5	26		51	0	0
		1×10-4	35		70	22	0
	paclitaxel	1×10-6	-		-	-	67
	Mitomycin-c	1×10-6	59		-	-	-
	5-Fluorouracil	2×10-5	-		74	69	-

 

 

The plates were washed five times with distilled water to remove trichloroacetic acid, growth medium low molecular weight metabolites, serum proteins etc and air-dried. The plates were stained with sulforhodamine B dye (0.4 % in 1% acetic acid, 100µl) for 30 minutes. The plates were washed five times with 1% acetic acid and then air-dried⁽⁷⁾. The adsorbed dye was dissolved in Tris-HCl Buffer (100μL, 0.01M, pH 10.4) and plates were gently stirred for 10 minutes on a mechanical stirrer. The optical density (OD) was recorded on ELISA reader at 540 nm. The cell growth was determined by subtracting mean OD value of respective blank from the mean OD value of experimental set. Percent growth in presence of test material was calculated considering the growth in absence of any test material as 100% and in turn percent growth inhibition in presence of test material was calculated.

 

ACKNOWLEDGMENT:

The authors are grateful to ISF College of Pharmacy, Moga for providing research facilities; and IIIM, Jammu for spectral analysis and pharmacological activity.

 

REFERENCES

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5.       Ducki S., Mackenzie G., dreedy B., Bioorganic and medicinal chemistry 17 (2009) 7711- 7722.

6.       Monks A., Scudiero D., Skehan P., Shoemaker R., Paull K., Vistica D., Hose C., Langley J., Cronise p, Vaigo-Wolfe A., Gray-Goodrich M., Campbell H., Boyd M., j Natl Cancer Inst 83 (1991) 757-766.

7.       Skehan P., Storeng R., Scudiero D., Monks A., McMahon J., Vistica D., Warren J., Bokesch H., Kenny S., Boyd M.,  J Natl Cancer Inst 82 (1990) 1107-1112.

 

 

Received on 25.03.2011        Modified on 05.04.2011

Accepted on 11.04.2011        © AJRC All right reserved