Determination of Antiproliferative Activity

The antiproliferative activity of the synthesised compoun determ -positive MCF-7 mammary carcinoma cell line. his assa med using estradiol as growth stimulating hormone in a nearly physiological concentration of 1nM. The

monopheno-lic d tive wer t consider r this evaluatio e to the

redu indi r cor onding di s.

The poun mple alkyl substituents showed no inhibitory effect on cell growth (data not shown). In contrast, the compounds carrying a long functionalised side chain demonstra endent grow nhibition. IC50-values from s assay are listed in table C18. The antiproliferative effects of the 2,5-diphenylfurans were mainly ds were ined in the ER T y was perfor

eriva s 48-50 of 2,5-diphenylfurans e no ed fo n du ced b ng affinities compared to thei resp phenol

com ds 43b-d of the series with si

ted a dose-dep th i The thi

influenced by the structure of the side chain. T rongest in ry effect with

of 22nM and 53nM, respectively, was found for compounds and 44d with the bifunc-tion e chain. F s d the pyrrol ohexyl ser antiprolifera activity was reduced by one or two order of magnitude. Compound 46b 50 -value >10µM).

he st hibito IC50-values 44c

al sid or the ulfone an idin ies the tive

proved to be inactive (IC

O

Table C18: Antiproliferative effects of 3,4-dialkyl-2,5-phenylfurans with functionalised side chains in MCF-7 and MDA-MB 231 breast cancer cells

order to exclude a non-specific cytotoxic action all 2,5-diphenylfurans were tested in hor-mone-independent MDA-MB 231 breast cancer cells. Their growth was not inhibited by these agents in concentrations up to 1µM. However, at higher concentrations all compounds exerted cytotoxic effects, but the IC50-values in the hormone-independent cell line exceeded the one in the MCF-7 cells at least by the factor of 10. The only exceptions were the compounds 45b In

and 45c. In fact, 45b was equally active in both cell lines what makes an non-specific action f this derivative likely (cf. table C18).

.2.3 Determination of Estrogenic and Antiestrogenic Activity in vitro

he dipheno lfurans were tested for their estrogenic and antiestro-enic activity in the luciferase assay using hormone-dependent MCF-7/2a cells that have been

Figure C19: Antiestrogenic effects of 3,4-dialkyl-2,5-diphenylfurans with functionalised side chains in MCF-7/2a cells

The simple alkyl derivatives 43b-d of 2,5-diphenylfurans showed no antiestrogenic effects at a concentration of 1µM (data not shown). All compounds with an aliphatic side chain, except 46b, ted the E2 ion in a do pendent manner (cf. figure

C19). The lowest IC , respective were obtaine e

com-pound and 44d in. Their an rogenic activ by one

order of magnitude lower than that of fulvestrant. All other compounds 45b-c and 47b-d in-hibited gene transcription with IC50-values that were by one or two orders of magnitude o

2

lic 3,4-dialkyl-2,5-dipheny T

g

stably transfected with a luciferase reporter gene under the control of an ERE. The antiestro-genic activity was determined by simultaneous treatment of the cells with 1nM E2 and the respective 2,5-diphenylfuran in various concentration.

-20 0

Luciferase activity (% of E2)

ICI

Luciferase activity (% of E2)

ICI

inhibi stimulated luciferase express se-de

50-values of 50nM and 67nM ly, d for th s 44c with the bifunctional side cha tiest ity was

higher. The ethyl derivatives were by a factor of 10 more potent than the corresponding methyl derivatives in each series of com 50-values (cf. table C20) were in good accordance with those from the chemosensitivity assay in MCF-7 cells, but there was no ob-vious correlation between the potencies in the luciferase assay and the RBA of these agents for ER possible expla rence in the experimental conditions of the two assay binding assay a cell-free syste whereas the transactivation assay required intact cells.

pounds. All IC

Table C20: Antiestrogenic activity of 3,4-dialkyl-2,5-phenylfurans with functionalised side chains in MCF-7/2a cells

he estrogenic potency of all test compounds was de d sim r ass cen-ation of 1µM in the absence of estradiol. At this con atio e s le alkyl derivatives 3b-d produced full estrogenic response with exception of com nd which bound only eakly to the ER (cf. table C21 left). For an exact dete atio f th trogen tivity of

T termine in a ila ay at a con

tr centr n th imp

4 pou 43b,

w rmin n o e es ic ac

these compounds dose-response curves were determined. The EC50-values showed an

in-right). This

crease in agonistic potency by the factor of 10 with increasing chain length (cf. table C21 behaviour did not correlate with the binding affinities of these agonist.

Table C21: Estrogenic activity of the 3,4-dialkyl-2,5-phenylfurans 43b-d in MCF-7/2a cells

ctrl = control; left: luciferase activity measured at a compound concentration of 1µM without E2 stimulation; right: Dose-response curves without E2 stimulation and the corresponding EC50 -values

he 2,5-diphenylfurans with functionalised side chains showed no agonistic activity at a con-entration of 1µM. All values except that of compound 46b were below that of the control ells. Luciferase activity below baseline levels are characteristic for potent antiestrogens and dicate the blockade of AF-1 mediated ligand-independent activation of the ER, that is re-ponsible for the basal luciferase activity in the control cells. Thus, 46b cannot be character-ed as an antiestrogen, which is in accordance with the low binding affinity and proliferative ctivity.

or an estimation of the residual estrogenic activity of these agents the levels of luciferase xpression were compared with those of the partial antiestrogen 4-hydroxytamoxifen and the ure antiestrogen fulvestrant (cf. figure C22). The compounds 44b-d with the bifunctional ide chain can be characterised as pure antiestrogens with a suppression of luciferase expres-T

ctrl E2 43b 43c 43d

60 80 100

Luciferase activity (% of E2).

120

Luciferase activity (% of E2)

43b

sion comparable to that of fulvestrant. The relative luciferase activities of compounds 45b and 5c with the pyrrolidinohexyl side chain exceed that of 4-hydroxytamoxifen and confirm the observation that the length of the side chain is . Contrary to revious studies with sulfones based on different core structures the sulfones 47b-d did not

ach the level of fulvestrant. In this respect they resemble 4-hydroxytamoxifen.

ination of estrogenic and antiestrogenic (antiuterotrophic) activity of test compounds in vivo. In vi-tro, compound 44b was shown to be a potent antiestrogen without any residual estrogenic

ac r this it w be esen le

the uterine w t. pro nfor th of

2,5-diphenylfuran-based antiestrogens in a mam lian organism ost of the non-steroidal pure antiestrogens suf

The drug was adm g/kg body weight, respectively, either

alone or in combination with estradiol. At the higher concentration the antiestrogen sup-pressed the estradiol stimulated uterus growth by 74% and demonstrated no significant estro-4

an important factor for antagonism p

re

Figure C22: Suppression of basal luciferase activity by 3,4-dialkyl-2,5-diphenylfurans with functionalised side chains in MCF-7/2a cells

The value for estradiol at a concentration of 1nM was set to 100% (not shown).

ctrl = control

2.2.4 Determination of Estrogenic and Antiestrogenic Activity in vivo