Biological Characterisation of the Benzopyran(one)s

5.2.1 Determination of Affinity and Selectivity for the ER

The synthesised benzopyrans and benzopyranones were tested for their binding affinities to the ER and their selectivity for one of the two ER subtypes. The binding curves of all test compounds were comparable in shape to the binding curve of E2, but shifted to higher con-centrations, which indicates a competitive displacement of the physiological ligand from the receptor binding site. The RBA values were determined by two methods using either calf

uterus cytosol as receptor source or the recombinant human ERα and ERβ. The RBA values shown in table C71 are the means of two or three independent experiments.

O

Table C71: Relative binding affinities of the benzopyran(one)s

All the benzopyrans and benzopyranones substituted with a long functionalised side chain in position C-4 demonstrated a slight preference for the ERα. In contrast, the unsubstituted ben-zopyran 139 showed a 8-fold selectivity for ERβ due to a rather low binding affinity to ERα.

The methylene and the carbonyl group in position 2, which is the only structural difference between the benzopyrans and benzopyranones, weakly affect the affinity and subtype selectivity of the compounds. The lipophilic methylene group favours the interaction with amino acid residues in both receptor subtypes more than the polar carbonyl function, as it can

concluded from the higher binding affinities of the benzopyrans. Contrary to the binding af-finities, the selectivities of the benzopyrans were somewhat lower than those of the corre-sponding benzopyranones. This finding reflects, that the carbonyl group rather than the me-thylene group contributes to ERα selectivity, because ligand binding pocket of ERα tolerates the carbonyl function better than ERβ.

The benzopyranone 127 with the bifunctional side chain displayed the highest selectivity for ERα (4.3-fold). The highest binding affinities in both series were found for the compounds bearing the bifunctional side chain with a basic nitrogen in position 7, which probably forms a hydrogen bond to ERα Asp351 or ERβ Asp303. A comparison of the binding affinities of the mono- and diphenolic derivatives 146 and 147 in the benzopyran series indicates that both hydroxy functions are involved in hydrogen bonding to the glutamate, arginine and histidine, respectively, of ERα and ERβ. The binding data of the corresponding benzopyranones 126 and 127 allow no definite statement in this context. The sulfones 129 and 141 in each series of compounds bound stronger to the ER than the corresponding sulfides 128 and 138.

The indene 135 displayed a 1.5-fold selectivity for ERβ, but the binding affinities to both ER subtypes in comparison to the structurally related 6-hyroxybenzofurans and 6-hydroxybenzo-thiophenes (cf. table C49 and C56) were much lower.

5.2.2 Determination of Antiproliferative Activity

The antiproliferative activities of the synthesised 3-phenylbenzopyrans and -benzopyranones were determined in the ER-positive MCF-7 mammary carcinoma cell line. The assay was performed using estradiol as growth stimulating hormone in a nearly physiological concentra-tion of 1nM.

All the compounds equipped with long functionalised side chains inhibited the cellular growth of these cells with IC50-values within a range of 30 and 500nM (cf. table C72). The sulfones 129 and 141 displayed equal activities, which can be rationalised by the similar RBA values.

The antiproliferative effect of the corresponding sulfides 128 and 138 was lower by a factor of ten. The benzopyran 147 that bound with the highest affinity to the ER exerted the strong-est inhibitory effect in MCF-7 cells with an IC50-value of 30nM. Its monophenolic derivatives 146 was slightly less active, which was in good accordance with the data from the binding assay. Surprisingly, the activities of the benzopyranones 126 and 127 with the bifunctional side chain were reduced by one order of magnitude in comparison to the corresponding

ben-zopyrans 146 and 147, although the binding affinities were similar. This discrepancy might be due to the different experimental conditions under which these assays were performed: cell-free conditions for the binding assay and intact cells for the proliferation assay.

O O

H

R² R¹

X

Comp. X R¹ R² MCF-7

IC50 [µM]

MDA-MB 231 IC50 [µM]

126 O (CH2)6N(CH3)(CH2)3SC5H11 H 0.2 1.6

127 O (CH2)6N(CH3)(CH2)3SC5H11 OH 0.2 1.6

128 O (CH2)10SC5H11 OH 0.4 6.0

129 O (CH2)10SO2C5H11 OH 0.05 5.6

138 H, H (CH2)10SC5H11 OH 0.5 6.2

141 H, H (CH2)10SO2C5H11 OH 0.06 1.9

146 H, H (CH2)6N(CH3)(CH2)3SC5H11 H 0.08 2.1 147 H, H (CH2)6N(CH3)(CH2)3SC5H11 OH 0.03 2.0

139 H, H H OH 1.5 1.4

135

O H

OH

OH > 10 > 10

ICI 0.004 >10

Table C72: Antiproliferative effects of the benzopyran(one)s in MCF-7 and MDA-MB 231 breast cancer cells

The benzopyran 139 without a substituent in position C-4 showed an inhibition of MCF-7 cell growth, which is very likely caused by non-specific cytotoxic effects of this compound, be-cause similar activity was observed in the assay with ER-negative MDA-MB 231 cells. Gen-erally, the inhibitory effect of the benzopyran(one)s with functionalised side chains on these hormone-independent MDA-MB 231 cells was at least by a factor of 10 smaller than that on the MCF-7 cells, which makes a non-specific drug action of these agents unlikely. The indene

135 showed no inhibition of cell proliferation in both cell lines up to a concentration of 10µM.

5.2.3 Determination of Estrogenic and Antiestrogenic Activity

The 3-phenylbenzopyrans and -benzopyranones with a long functionalised side chain were tested for their estrogenic and antiestrogenic activity in the luciferase assay using the ER-positive MCF-7/2a cells The antiestrogenic activity was determined by simultaneous treat-ment of these cells with 1nM E2 and the respective benzopyran(one) in various concentration.

The IC50-values calculated from the dose-response curves were similar to those obtained from the chemosensitivity assay with MCF-7 cells (cf. table C73), except for the monophenolic benzopyran 146, whose inhibitory effect on E2-stimulated luciferase expression was by a fac-tor of 2 higher and, thus, in the same range as the diphenolic derivative 147. Compound 147 displayed the strongest antiestrogenic effect, which was by a factor of 10 lower than that of the steroidal antiestrogen fulvestrant.

O O

H

R² R¹

X

Comp. X R¹ R² IC50 [µM]

126 O (CH2)6N(CH3)(CH2)3SC5H11 H 0.2

127 O (CH2)6N(CH3)(CH2)3SC5H11 OH 0.2

128 O (CH2)10SC5H11 OH 0.4

129 O (CH2)10SO2C5H11 OH 0.05

138 H, H (CH2)10SC5H11 OH 0.3

141 H, H (CH2)10SO2C5H11 OH 0.06

146 H, H (CH2)6N(CH3)(CH2)3SC5H11 H 0.04

147 H, H (CH2)6N(CH3)(CH2)3SC5H11 OH 0.03

ICI 0.003

Table C73: Antiestrogenic activity of the benzopyran(one)s in MCF-7/2a cells

The estrogenic potency of the benzopyran(one)s was determined in a similar assay at a con-centration of 1µM in the absence of estradiol. At this concon-centration none of the compounds tested showed agonistic activity. The luciferase expression levels were below that of the con-trol cells (cf. figure C74), what is characteristic for potent antiestrogens. It indicates the blockade of a ligand-independent activation of the ER, that is responsible for the basal luciferase activity in the control cells. For a better estimation of the residual estrogenic activ-ity the levels of luciferase expression were compared with those of the partial antiestrogen 4-hydroxytamoxifen and the pure antiestrogen fulvestrant. All the tested benzopyrans and benzopyranones, except the compounds 126 and 146, suppressed luciferase expression to the same extent as fulvestrant did. The two monophenols 126 and 146 were also devoid of agonist activity though their suppression was somewhat weaker, but still better than that of the partial antiestrogen 4-hydroxytamoxifen.

0 2 4 6 8 10 12 14 16

ctrl 126 127 128 129 138 141 146 147 ICI OH-tam

Luciferase activity (% of E2)

Figure C74: Suppression of basal luciferase activity by benzopyran(one)s with functionalised side chains in MCF-7/2a cells

The value for estradiol at a concentration of 1nM was set to 100%.

ctrl = control

5.3 Conclusion

It was possible to introduce aliphatic side chains with various functional groups into position C-4 of 3-phenylbenzopyrans and –benzopyranones. All the synthesised compounds were characterised as potent estrogen antagonists without residual estrogenic activity. They showed

a preference for ERα contrary to antiestrogens based on the 2-phenylbenzo[b]furan, whose heterocyclic core is by one carbon atom smaller. The antiproliferative and antiestrogenic po-tency of the 3-phenylbenzopyran(one)s was by one order of magnitude higher than that of the corresponding 2-phenylbenzofurans. In comparison to the 2,5-diphenylfurans the selectivity of the 3-phenylbenzopyran(one)s for ERα was slightly reduced. The antagonistic potency of the benzopyran 146 with the bifunctional side chain was comparable to that of the corre-sponding 2,5-diphenylfurans, whereas that of the benzopyranones was by one order of magni-tude smaller. However, in both series of benzopyrans and benzopyranones the sulfones and sulfides were by a factor of 10 more active than in the 2,5-diphenylfuran series, which was in good accordance with the higher binding affinities. Compound 129 with the sulfonyl group in the side chain was the most active in the class of antiestrogens based on the 3-phenyl-benzopyranone scaffold.