Chem. Eur. J. 2003, 9, 5223 ± 5236 ferrocen.pdf

FULL PAPER

Synthesis, Biochemical Properties and Molecular Modelling Studies of

Organometallic Specific Estrogen Receptor Modulators (SERMs), the

Ferrocifens and Hydroxyferrocifens :Evidence for an Antiproliferative Effect

of Hydroxyferrocifens on both Hormone-Dependent and

Hormone-Independent Breast Cancer Cell Lines

Siden Top,* [a] Anne Vessi ¡ res, [a]

Guy Leclercq, [b]

Jacques Quivy, [b]

J. Tang, [a]

J. Vaissermann, [c] Michel Huch ¬, [a] and G ¬ rard Jaouen* [a]

Abstract: A series of ferrocene deriva-

tives based upon the structure of the

antiestrogenic drug tamoxifen or of its

activemetabolitehydroxytamoxifenhas

been prepared and named by analogy

ferrocifens and hydroxyferrocifens. This

series includes 1-[4-(O(CH 2 ) n NMe 2 )-

phenyl]-1-phenyl-2-ferrocenyl-but-1-ene

and 1-[4-( O(CH 2 ) n NMe 2 )phenyl]-1-(4-

hydroxyphenyl)-2-ferrocenyl-but-1-ene,

with n

the isomers was obtained within one

hourinchloroform.TheX-raystructure

of ( E )-1-[4-( O(CH 2 ) 2 NMe 2 )phenyl]-1-

(4-hydroxyphenyl)-2-ferrocenyl-but-1-ene

hasbeen determined. Therelative bind-

ingaffinity(RBA)valuesofthehydrox-

yferrocifens for the estrogen receptor

alpha(ER )wasgoodtomoderate,with

valuesdecreasingprogressively withthe

length of the basic chain. The RBA

values found for the estrogen receptor

beta (ER ) are equal to or slightly less

thanthosefoundforthealphaform.The

lipophilicity of the hydroxyferrocifens

are superior to the values found for

estradiol and increase with lengthening

of the chain. The antiproliferative ef-

fectsofthefourhydroxyferrocifenswith

n 2, 3, 5 and 8 were studied on four

breast cancer cell lines (MCF7, MDA-

MB231, RTx6 and TD5) possessing

different levels of ER . On MCF7 cells

containing high levels of ER ,hydroxy-

ferrocifensbehaveasantiestrogens.Ata

molarity of 1

the effect is close to

that of hydroxytamoxifen (used for

reference) when n

2 or 5, more

marked when n

3, and weaker when

n 8.Ferrocenealonehasnoeffect.For

the MDA-MB231 cells, classed as a

hormone-independentbreastcancercell

line, on the other hand, the hydroxyfer-

rocifens show remarkable antiprolifera-

tivebehaviourwhilethe hydroxytamox-

ifen is completely inactive. Hydroxyfer-

rocifens therefore show the unique

property of being active both on hor-

mone-dependent and on hormone-inde-

pendent breast cancer cell lines. The

molecular modelling study provides

some clues for understanding of the

antagonist effect of these molecules,

while an additional cytotoxic effect due

to the vectorised ferrocenyl unit is

revealed in some occasions.

2, 3, 5 and 8, and 1-[4-

( O(CH 2 ) 2 NMe 2 )phenyl]-1-(4-hydroxy-

phenyl)-2-ferrocenylethene. Most of

these molecules have been synthesised

by McMurry cross-coupling of the ap-

propriateketones,exceptfortheethene

complexes, which were prepared by a

four-step reaction sequence starting

fromtheferrocenylaceticacid.Allthese

compounds were obtained as mixtures

of Z and E isomers. The isomers were

separated in the cases of the ferrocenyl

derivativesoftamoxifenand hydroxyta-

moxifen( n 2).Noisomerisationofthe

Z and E isomers occurred in DMSO

after one day, while a 50:50 mixture of

Keywords: antitumor agents ¥ bio-

organometallic chemistry ¥ breast

cancer ¥ iron ¥ tamoxifen

[a] Dr.S.Top,Prof.G.Jaouen,Dr.A.Vessie¡res,Dr.J.Tang,Dr.M.Huche¬

Laboratoire de Chimie Organome ¬ tallique

UMR CNRS 7576

Ecole Nationale Supe ¬ rieure de Chimie de Paris

11, rue Pierre et Marie Curie

75231 Paris 05 (France)

Fax: (

Institut Jules Bordet, Rue He¬ger Bodet, 1

1000-Bruxelles (Belgium)

[c] Dr. J. Vaissermann

Laboratoire de Chimie des Me¬taux de Transition

Universite ¬ Pierre et Marie Curie

75230 Paris Cedex 05 (France)

SupportinginformationforthisarticleisavailableontheWWWunder

http://www.chemeurj.org or from the author, including TableS2 (frac-

tional coordinates), TableS3 (interatomic distances), Table S4 (bond

angles) and a listing of structure factors for ( E )-3a.

33)1-43-26-00-61

E-mail:gerard-jaouen@enscp.jussieu.fr

[b] Prof. G. Leclercq, Dr. J. Quivy

Laboratoire de Cance ¬ rologie Mammaire

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DOI:10.1002/chem.200305024 ¹ 2003 Wiley-VCHVerlag GmbH&Co. KGaA, Weinheim

Chem. Eur. J. 2003, 9 , 5223±5236

FULL PAPER

S. Top, G. Jaouen et al.

Introduction

because the molecule isomerizes in solution, it is the mixture

thatisadministered.Thismayexplainacertain dualityinthe

effects of tamoxifen, but it has not proved a barrier to its

success.

The hydroxy group is essential in the active metabolite of

thismolecule,sinceitconfersanincreasedlevelofaffinityfor

the estrogen receptor.As is well known, this receptor plays a

key role in the proliferation of hormone-dependent tumours

and is one of the most important target molecules for

tamoxifen, although other targets do exist. [16]

Considerable interest has been generated by coordination

compoundsofplatinum,andsoastrategyhasbeendeveloped

based on coupling of platinum, with its known efficacy in

chemotherapy and high general cytotoxicity, to various

ligands of the estrogen receptor through a nitrogen atom,

with the aim of creating antiestrogenic compounds that are

able both to target the binding site and to provide increased

cytotoxicity. [17±21] Unfortunately the Pt N coordination bond

inthesecomplexesistooweakanditappearsthatthemetalis

unable to reach the target because it hydrolyses too quickly

and is too bulky in size. This is the limiting factor in the

appealing strategy of coupling a transporter group to a

coordination complex in order to increase its cytotoxicity.

However,thisconceptoftheantiestrogenictransportercan

be revisited from another perspective. Instead of compounds

withcoordinationbonds,whichareinherentlyquiteweak,we

mayenvisagetheuseofspecies

with strong metal±carbon co-

valentbonds,andhereweenter

the field of organometallic

chemistry. Metallocenes are in

fact known to have their own

antitumor properties, based on

a different mechanism from

that of the cisplatin com-

plexes. [22] To our knowledge,

however, previous to our own

work they have not been incorporated into an antiestrogenic

entity. The idea of attempting this, as a means towards the

discovery of antiestrogens with improved cytotoxic proper-

ties, was appealing.

Ferrocene(

Tamoxifen is currently the most widely used antiestrogen in

adjuvant therapy of hormone-dependent breast cancers. Its

active metabolite is 4-hydroxy-tamoxifen. [1±3]

Since tamoxifen is active only against tumours that are

estrogen receptor positive (ER ), and frequently gives rise

to resistance after prolonged use, the search for related but

different agentshasintensifiedconsiderablyoverthelast few

years. [4±8]

Attention quickly centredon variationson the structure of

the triphenylethylene skeleton, resulting in new specific

estrogen receptor modulators (SERMs) that include toremi-

fene, droloxifene and idoxifene [7, 9] .

Derivatives of 2-phenylindoles have also been developed,

giving zindoxifene, and also from aryl-benzo[b]thiophenes,

which provide access to raloxifene and thence to steroidal

compoundsmodifiedbytheadditionoflongcarbonchains(of

aroundninecarbonatoms),theselastcompoundsbehavingas

pure antiestrogens. [3, 10] It should be noted that all known

SERMs bear a chain of varied length but always with p or

electron sites (e.g., NR 2 , S R, SO R, SO 2 R etc.).

It recently became possible to rationalise this observation

whentheX-raycrystalstructureoftheligand-bindingdomain

(LBD) of estrogen receptor alpha was elucidated. [11±15] This

study showed that the bioligand can bind as an antagonist,

creating a new pocket in a flexible area of the protein and

allowing accommodation of the basic chain. The result of

these conformational effects is to modify the position of

helix12 of the receptor, preventing it from interacting with

certain effectors present in the target cell and therefore from

carrying out its function as an activator of specific genes.

Tamoxifen acts in vivo as a particularly well tolerated

cytostatic agent. It should be noted that the molecule exists

both in Z and in E configurations, of which the Z isomer has

been shown to be the most strongly antiestrogenic, but

5 -C 5 H 5 ) 2 Feis thearchetypalmetallocene.One

could envisage using it to increase the cytotoxicity of

hydroxytamoxifen by attaching this moiety to the key

skeleton of diphenylethylene bearing the crucial hydroxy

anddimethylaminogroups.Infact,aferrocenylmoietycould

presentanumberofadvantages.Ferroceneisinherentlymore

aromatic than benzene, so it is likely that replacement of the

phenyl nucleus of 4-hydroxy-tamoxifen would be well

justified;itischemicallyfairlystableinvariousnon-oxidizing

mediaandhasbeenreportedtohaveantitumoractivitydueto

metabolic formation of ferricinium ions in situ. [23] Other

metallocenes with metals such as Ti, [24] V, Mo, Re, [25] Co and

Rumayalsobeenvisaged.Totestthevalidityoftheconcept,

we synthesised derivatives of ferrocene following the base

structure of tamoxifen, in which the aromatic

ring is

replaced by a ferrocenyl group and the dimethylamino side

chain is of variable length. Byanalogy these derivatives were

named ferrocifens and hydroxyferrocifens.

5224

¹ 2003 Wiley-VCHVerlag GmbH&Co. KGaA, Weinheim www.chemeurj.org

Chem. Eur. J. 2003, 9 , 5223±5236

Ferroci f ens and Hydroxyferrocifens

5223±5236

Preliminary studies revealed that the length of the chain

appeared to have a considerable effect on the biological

properties of the complexes. [26, 27] Here we present the syn-

thesis, molecular modelling studies and biological properties

of a series of ferrocifens and hydroxyferrocifens.

Results and Discussion

Synthesis of the products: The first hydroxyferrocifens were

prepared in our laboratory by starting from ethyl ferroceny-

lacetate and using a long, painstaking and impractical

method. [28] After testing several synthetic routes, we found

that a route based on McMurry coupling (Zn, TiCl 4 ) was the

most convenient, since it is shorter when starting from

metallocene ketones. [29] It is easy to perform and gives good

yields of mixtures of Z and E isomers. This method has since

been extended to all the complexes described here. Two

syntheticstrategieswereadopted,dependingonthecommer-

cial availability of the particular reagent X(CH 2 ) n NMe 2 .In

cases in which the reagent is available (i.e., for n

Scheme 1. Synthesis of ferrocifens and hydroxyferrocifens with n

2or3

by McMurry cross-coupling.

2 and 3),

the synthesis begins with a coupling reaction between

propionylferrocene, obtained by alkylation of the ferrocene

by means of a Friedel±Crafts reaction, and hydroxybenzo-

phenone or dihydroxybenzophenone (Scheme1).

After the propionylferrocene/dihydroxybenzophenone

mixture has been stirred in the presence of the McMurry

reagent, the complex 1 resulting from the cross coupling is

obtained as the major product with a yield of 52%. [26]

Coupling with hydroxybenzophenone gives 2 as a mixture of

two isomers ( Z and E ). [29] Heating of these two compounds

withsodiumhydroxideinacetone,followedbyadditionofthe

chloroalkyldimethylaminederivative,givesthehydroxyferro-

cifens 3a and 3b and ferrocifens 4a and 4b in yields ranging

between 26% and 66%. Alkylation of 1 similarly gives the

dialkylation compounds 5aand 5b.

In cases in which the reagent giving access to the amine

chain X(CH 2 ) n NMe 2 is not commercially available (i.e., for

Scheme 2. Synthesis of ketones 6c, 6d and 7d, bearing amino chains

O(CH 2 ) n X( n

5, 8).

dimethylamine hydrochloride in an autoclave. This gives the

hydroxyferrocifens 3c and 3d and the ferrocifens 4c and 4d.

Forcomplexes 14 and 15,inwhich ahydrogen replaces the

ethyl group, the synthesis is performed from ferrocenylacetic

acid (11) by the route shown in Scheme4.

Theacid 11 isfirstconvertedintotheester 12.Treatmentof

12 with methoxyphenyllithium gives an alcohol intermediate,

which dehydrates to give 1,1-bis(4-methoxyphenyl)-2-ferro-

cenylethene (13). Demethylation of 13, to give the diphenol

14, is then induced by treatment with BBr 3 in dichloro-

methane. The final step is to attach the dimethylamino chain

onto one of the two phenolic functions of 14. Use of sodium

ethanolate followed by 2-chloroethyl-dimethylamine hydro-

chloride gives a mixture of the two isomers ( Z E )of15 in

41% yield for this final step. The secondary dialkylated

compound 16 is also isolated (30%).

5 and 8), it is necessary to start with alkylation of the

mono- or dihydroxybenzophenone by an , -dihaloalkane

(Scheme2). This gives the monoalkylated derivatives 6c, 6d

and 7d, as well as the dialkylated derivative 8 (in the case of

dihydroxybenzophenone).

The obtained ketones are then coupled with propionylfer-

rocene in the presence of the McMurry reagent to give the

halogenated compounds 9c, 9d, 10 c and 10 d (Scheme3). In

the final step, these derivatives are allowed to react with

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Chem. Eur. J. 2003, 9 , 5223±5236

FULL PAPER

S. Top, G. Jaouen et al.

hydroxyferrocifen 3a, separation was

achieved by fractional crystallisation

from an ethyl ether/hexane mixture.

Inthecaseofferrocifen 4a,the Z and

E isomers were separated by plate

chromatography after several treat-

ments with a toluene/pyridine (6:1)

eluent. Separation of the other iso-

mers ( n

3, 5 and 8) by these meth-

ods was not possible.

Structural study: Identification of the

structuresofthe Z and E isomerscan

only be accurately determined by

X-ray crystallographic study. After

separation of the Z and E isomers,

the complexes 3a were allowed to

crystallise from diethyl ether/hexane

(5:1) mixtures. Only the E isomer of

3a gave crystals of sufficient quality

to perform an X-ray structural anal-

ysis.Thecrystalsweremonoclinicand

belonged to the P 2 1 / c space group. The crystallographic data

are given in Table1, and the ORTEP diagram of ( E )-3a is

shown in Figure1. To facilitate comparisons between the

structuresof ferrocifen 4aand hydroxyferrocifen 3awehave

alsoincludedtheORTEPdiagramfor( Z )-4a,thestructureof

which was published in an earlier paper. [29]

Scheme 3. Synthesis of ferrocifens and hydroxyferrocifens with n

5 or 8 by McMurry cross-coupling.

Table 1. Summary of crystallographic data for ( E )-3a.

formula

C 30 H 33 O 2 NFe

495.4

a [ä]

15.174 (4)

b [ä]

13.259 (4)

c [ä]

14.197 (18)

[

]

112.17 (1)

V [ä 3 ]

2645(1)

crystal system

monoclinic

space group

P 2 1 / c

F (000)

1048

linear absorption coefficient

[cm 1 ] 6.56

[gcm 3 ]

1.24

diffractometer

Enraf±Nonius CAD4

radiation

Mo K (0.71070ä)

scan type

scan range (

)

0.9

0.34 tg

) 4±50

temperature of measurement room temperature

octants collected 0,8; 0,18 ; 0,22

no. of data collected 4579

no. of unique data used for refinement 1034 ( I 3

Limits (

( I ))

F o

F c

F o

0.053

Rw [a]

{

w (

F o

F c

) 2 /

wF o 2 } 1/2

0.057

absorption correction

DIFABS (min

0.90, max

1.16 )

extinction parameter

none

goodness of fit s

1.17

Scheme 4. Synthesis of hydroxyferrocifen 15 from ferrocenylacetic acid

(11).

no. of variables

149

min [eä 3 ]

0.47

max [eä 3 ]

0.62

All the ferrocifens and hydroxyferrocifens are obtained as

mixtures of Z and E isomers. It was possible to separate the

isomersinthecasesinwhich n 2(complexes 3aand 4a).For

[a] w

((

F o

F c

)/6

( F o )) 2 ] 2 with w

rA r T r (X) with three

coefficients3.79,

0.810and2.74 foraChebyshevSeries,forwhichXis F c /

F c (max).

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¹ 2003 Wiley-VCHVerlag GmbH&Co. KGaA, Weinheim www.chemeurj.org

Chem. Eur. J. 2003, 9 , 5223±5236

Ferroci f ens and Hydroxyferrocifens

5223±5236

conformation,withtheironatom

bound symmetrically to the two

cyclopentadienyl rings, with an

averageC Fedistanceof2.04ä.

A slight deformation is observed

at the level of the ethylene skel-

eton. The angle formed by the

double bond and one of the four

substituents is of the order of

122±124 for both the cis - and

the trans -tamoxifens. In the case

of ( E )-3a, the bond angles have

thefollowingvalues:C(11)-C(1)-

C(2): 126.2 , C(1)-C(2)-C(31):

124.6

, C(4)-C(1)-C(2): 119.0

and C(1)-C(2)-C(21): 120.9

.In

the case of ( Z )-4a, the bond

angleshavethefollowingvalues:

C(11)-C(1)-C(2): 129.0

, C(1)-

C(2)-C(31): 125.0

, C(3)-C(1)-

C(2): 114.4 , and C(1)-C(2)-

C(21): 122.0 . There is thus a

widening of the angle at the side

at which the ferrocenyl group is

located, and a narrowing on the

ethyl side. This is probably at-

tributable to the greater bulk of

the ferrocenyl group relative to

the phenyl. It can also be seen

that the four substituents on the

double bond do not lie in the

same plane, the C(11)-C(1)-

C(2)-C(31) atoms forming a di-

hedralangle of 13

in the case of

( E )-3a, while in ( Z )-4a they

form a dihedral angle of only

1.25 . The twist angle formed

between the plane of the aro-

matic ring and the plane of the

ethylene skeleton often attracts

theattentionofresearchers.This

anglehasbeenfoundtobeofthe

order of 52 to 55 for the three

rings of cis -tamoxifen, and sim-

ilar values have also been found

for trans -tamoxifen. In the case

of ( E )-3a, the C 6 H 4 OHring

forms an angle of 55 with the

plane of the double bond, in

agreement with those of ( Z )-

and ( E )-tamoxifen. In contrast,

the C 6 H 4 OCH 2 CH 2 NMe 2 ring

Figure 1. X-ray structures of ( Z )-4a and ( E )-3a.

The structures of ( E )-3a and ( Z )-4a may be compared to

those of cis - and trans -tamoxifen determined by Kilbourn [30]

and Precigoux, [31] respectively. The following points may be

noted: the interatomic distances in the central double bonds

(C(1) C(2)) are 1.36ä in( E )-3aand 1.37äin ( Z )-4a, while

thatin cis -tamoxifen( E )is1.33äandthatin trans -tamoxifen

( Z ) is 1.34ä. The ferrocenyl group appears in its normal

formsanangleof74

greaterthanthelistedvalue.

The distance between the O(1) and O(2) groups in ( E )-3a is

9.65ä. Finally, we note that the distance between O(3) and

O(17) is 10.9ä for estradiol [32] and 12.1ä for diethylstilbes-

trol (DES). [33] This X-ray analysis shows that the hydroxyfer-

rocifens have the size characteristics of a nanomediator for

the estrogen receptor.

,about20

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Chem. Eur. J. 2003, 9 , 5223±5236

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