Synthesis, binding and structure-affinity studies of new ligands for the microsomal anti-estrogen binding site (AEBS)

Article abstract of DOI:10.1016/S0968-0896(00)00119-X

New compounds have been synthesized based on the structure of the anti-tumoral drug tamoxifen and its diphenylmethane derivative, N,N-diethyl-2-[(4-phenyl-methyl)-phenoxy]-ethanamine, HCl (DPPE). These new compounds have no affinity for the estrogen receptor (ER) and bind with various affinity to the anti-estrogen binding site (AEBS). Compounds 2, 10, 12, 13, 20a, 20b, 23a, 23b, 29 exhibited 1.1-69.5 higher affinity than DPPE, and compounds 23a and 23b have 1.2 and 3.5 higher affinity than tamoxifen. Three-dimensional structure analysis, performed using the intersection of the van der Waals volume occupied by tamoxifen in its crystallographic state and the van der Waals volume of these new compounds in their calculated minimal energy conformation, correlated well with their pKi for AEBS (r=0.84, P<0.0001, n=18). This is the first structure-affinity relationship (SAR) ever reported for AEBS ligands. Moreover in this study we have reported the synthesis of new compounds of higher affinity than the lead compounds and that are highly specific for AEBS. Since these compounds do not bind ER they will be helpful to study AEBS mediated cytotoxicity. Moreover our study shows that our strategy is a new useful guide to design high affinity and selective ligands for AEBS. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00119-X

Bioorganic & Medicinal Chemistry 8 (2000) 2007±2016
Synthesis, Binding and Structure±Anity Studies of New Ligands
for the Microsomal Anti-Estrogen Binding Site (AEBS)
Marc Poirot,a,* Philippe De Medina,a Frederic Delarue,a Jean-Jacques Perie,b
Alain Klaebeb and Jean-Charles Fayea
aINSERM U 397, Institut Claudius Regaud, 20-24 rue du pont Saint-Pierre, 31052 Toulouse Cedex, France
b
Â
Groupe de Chimie Organique Biologique, SPCMIB, UMR CNRS 5068, Universite Paul Sabatier,
118 route de Narbonne, 31062 Toulouse Cedex, France
Received 14 March 2000; accepted 3 May 2000
AbstractÐNew compounds have been synthesized based on the structure of the anti-tumoral drug tamoxifen and its diphe-
nylmethane derivative, N,N-diethyl-2-[(4-phenyl-methyl)-phenoxy]-ethanamine, HCl (DPPE). These new compounds have no a-
nity for the estrogen receptor (ER) and bind with various anity to the anti-estrogen binding site (AEBS). Compounds 2, 10, 12,
13, 20a, 20b, 23a, 23b, 29 exhibited 1.1±69.5 higher anity than DPPE, and compounds 23a and 23b have 1.2 and 3.5 higher anity
than tamoxifen. Three-dimensional structure analysis, performed using the intersection of the van der Waals volume occupied by
tamoxifen in its crystallographic state and the van der Waals volume of these new compounds in their calculated minimal energy
conformation, correlated well with their pKi for AEBS (r=0.84, P<0.0001, n=18). This is the ®rst structure-anity relationship
(SAR) ever reported for AEBS ligands. Moreover in this study we have reported the synthesis of new compounds of higher anity
than the lead compounds and that are highly speci®c for AEBS. Since these compounds do not bind ER they will be helpful to
study AEBS mediated cytotoxicity. Moreover our study shows that our strategy is a new useful guide to design high anity and
selective ligands for AEBS. # 2000 Elsevier Science Ltd. All rights reserved.
Tamoxifen is a triphenylethylenic antiestrogen (Fig. 1a)
widely used around the world in the management and the
prevention of breast cancers.1 The rational for this appli-
cation was the ability of tamoxifen to block the mitogenic
activity of 17b-estradiol on the growth of breast cancer
cells. This involves the competition of tamoxifen with
estradiol on the estrogen receptor. However, tamoxifen
act as a partial agonist of estradiol and induces bene®cial
hypolipemic e€ects on the patient, but increases the risk
of development of endometrial cancer.1 This prompted
the development of pure antagonists, moreover, tamox-
ifen produced a cytotoxicity that is independent of ER2,3
that might involve other cellular targets such as Protein
kinase C,4 calmoduline,5 and a high anity microsomal
anti-estrogen binding site (AEBS).6,7 Interestingly the
development of ligands that have no anity for ER and
that are speci®c of AEBS has produced compounds of
potential clinical value since they are cytotoxic against
tumor cells8 10 and display antiviral activities.11 Such
compounds induced cytotoxicity in a dose and time
dependent way of tumoral cell lines of various ori-
gins9,10 but are only cytostatic on primary culture of
normal bovine endothelial aortic cells.12 In particular, a
diphenylmethane compound, the N,N-diethyl-2-[(4 -
phenyl-methyl)-phenoxy]-ethanamine HCl (DPPE) (Fig.
1b), was the ®rst speci®c and high anity ligand for
AEBS and no anity for ER.8 This compound has
recently been successful in the treatment of chemother-
apeutic refractive cancers in phase I and II clinical
trials,13 17 and is now into phase III trials. In vitro studies
have shown that the potentiation of the chemotherapeutic
index of cytotoxic drugs by tamoxifen and DPPE was
mediated by AEBS.18 Although DPPE's eciency is
moderate, it was tested on patients bearing di€erent
kinds of tumor independently of their hormonosensitiv-
ity (melanoma, prostate, ovarian. . .).13 17 Since the a-
nity of DPPE for AEBS was 20 times lower than that of
tamoxifen, it might be of particular interest to design
higher anity and selective ligands to target AEBS.
Structure±anity studies revealed that AEBS exhibits
high anity for two di€erent classes of compounds that
are competitive inhibitors of tamoxifen on AEBS. The ®rst
class includes arylaminoethyl compounds, they are catio-
nic and amphiphillic compounds such as triarylethylene,
AEBS: anti-estrogen binding site; ER: estrogen receptor; ATCC:
American tissue culture collection; PBS: phosphate bu€er saline;
EDTA: ethylene-diamine-tetra-acetic acid; FCS: fetal calf serum
*Corresponding author. E-mail: poirot@icr.fnclcc.fr
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00119-X

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M. Poirot et al. / Bioorg. Med. Chem. 8 (2000) 2007±2016
Figure 1. Chemical structure of AEBS ligands; (a) tamoxifen: (b) DPPE.
triarylpropenone, diarylbenzopyran, diarylbenzofuran
and diphenylmethanes.18,19 The second class includes
neutral synthetic oxysterol derivatives restricted to 6- and
7-ketocholestanol.20 Ketocholestanol, diarylbenzofuran
and diphenylmethane compounds bound to AEBS with
selectivity over ER, but the diphenylmethane series is the
only series that have been demonstrated not to a€ect
calmoduline,5,21 or PKC dependent events.22,23 In this
paper we reported the synthesis of new compounds
derived from the structure of the diphenylmethane
compound DPPE; their anity for AEBS and for the
estrogen receptor were measured, and a structure±anity
study was done.
Scheme 2. Synthesis of compounds 3±8, 11; RˆPhCH2.
compound 5 (Scheme 2). Reaction of compound 5 with
potassium phtalimide gave compound 6, and its hydrazi-
nolysis gave compound 7 (Scheme 2). Treatment of com-
pound
7 with 3,5-dimethylpyrazole-1-carboxamidine
nitrate in methanol resulted in compound 8 (Scheme 2).
Reaction of compound 2 in its free base state with
methyliodide gave compound 9 (Table 1). Treatment of
compound 5 with pyrrole gave compound 11 (Scheme
Results and Discussion
Synthesis
Table 1. Diphenylmethane derivatives of tamoxifen with altered
basicitya
We explored the structure±anity relationship of a series
aryloxyethyl derivatives based on the structure of a
tamoxifen derivative N,N-diethyl-2-[(4-phenyl-methyl)-
phenoxy]-ethanamine HCl (DPPE) (Fig. 2). Compounds
were synthesized according to Schemes 1 and 2.
Compound
R
pKa
Mp
(ꢀC)
Formula
Ki
(nM)
Compounds 1±2, 10±21b and 23a±25 were prepared in
high yield by reacting phenol with potassium carbonate as
a base with the appropriate side-chain aminoethylchloride
in a dimethylformamide:acetone (1:1) solution (Scheme
1). Compound 3 was similarly prepared and was subse-
quently transformed into the mesylate 4 by reaction of
the compound 3 with methanesulfonylchloride (Scheme
2). Treatment of compound 3 with triphenylphosphine
and tetrabromocarbon in methylenechloride produced
Tamoxifen
1 (DPPE)
2
3
4
5
N(CH3)2
10.8
Ð
2.5Æ0.5
N(CH2CH3)2 10.9 156±158 C19H25NO 55.6Æ2.2
N(CH3)2
OH
10.8 178±180 C17H21NO 42.3Æ3.1
Ð
Ð
Ð
70±72
58±60 C16H18O4S
60±62 C15H15OBr
C
15H16O4
N.M.
N.M.
N.M.
OSO2CH3
Br
6
Ð
80±82 C23H19NO3 N.M.
7
8
9
10
11
12
13
NH2
10.7 226±228 C15H18NOCl 924Æ18
NHC(ˆNH)NH2 13.65 102±104 C16H19N3O 1530Æ26
N(CH3)+3
c-N(C4H8)
c-N(C4H4)
10.8 138±140 C18H24NOI 114Æ5.1
11.3 170±172 C19H23NO
oil C18H19NO
8.8Æ0.9
Ð
N.M.
c-N(C5H10)
c-N(C4H8)O
11.2 180±182 C20H25NO 28.1Æ1.3
8.7 185±186 C19H23NO2 17.6Æ1.2
Figure 2. Structure±anity relationship for the diphenylmethane based
derivatives.
aAnalysis for C, H, and N are withinÆ0.4% of the theoretical values.
Rat liver membranes were incubated with 3 nM of [3H]tamoxifen and
12 concentrations of unlabeled test ligand ranging from 0.1 nM to
10 000 nM or 1 mM to 10 000 mM. Assays with [3H]tamoxifen include
1 mM of 17û-estradiol. IC50 values were determined using the iterative
curve ®tting program GraphPad prism. IC50 values were converted
into the apparent Ki using the Cheng±Pruso€ equation,30 and the Kd
values. Values are the average of three experiments, each carried out in
triplicate. N.M.=not measurable.
Scheme 1. Synthesis of compounds 1, 2, 10, 12±21b, 23a±25; RˆH,
CH3, Cl, Br, I, tBu, PhCH2, PhCO, PhO, PhCHˆCH, PhCH2CO;
R1ˆN(CH3)2, N(C2H5)2, c-N(C2H4)2, c-N(C2H4)CH2, c-N(C2H4)O.

M. Poirot et al. / Bioorg. Med. Chem. 8 (2000) 2007±2016
2009
2). Synthesis of compounds 27±29 has been reported
previously.4 Treatment of benzophenonic compounds
21a, 21b and 25 in CF3COOH using NaBH4 give
Ligand binding
24
The synthesized compounds were evaluated for their
binding anity for the estrogen receptor (ER) extracted
from MCF-7 cells by competing with [3H]estradiol, and
for binding to rat liver microsomal AEBS by competing
with [3H]tamoxifen. In this study tamoxifen displays a
Ki of 2.4 nM for the estrogen receptor (ER) whereas
DPPE and the new compounds prepared in this study
have no anity for ER. This indicates that the phenyl-
propene part of tamoxifen (Fig. 1) is required for estrogen
binding anity but not for binding to AEBS. We have
looked at the consequence of structural modi®cation of
DPPE (Fig. 2) on the anity for AEBS. The binding
anities for AEBS are expressed as Ki and are listed in
Table 1 to 4.
benzhydrol derivatives 22a, 22b and 26 (Table 3). Com-
pounds 30 to 32 were obtained by treatment of the ani-
line 28 in its free base state with the appropriated
anhydride in dioxane (Table 4).
Table 2. 1-(2-N-morpholinoethoxy)benzenesa
Compound
R
Mp, ꢀC
Formula
Ki (nM)
14
15
16
17
18
19
H
Cl
Br
189±190
222±224
180±182
178±180
208±210
190±192
C
C
12H17NO2
12H16ClNO2
N.M.
N.M.
3820Æ110
1630Æ47
N.M.
C12H16BrNO2
C12H16INO2
C13H19NO2
C16H25NO2
Importance of the protonated side chain in the
diphenylmethane series
I
CH3
tBu
248Æ18
Table 1 shows the consequences of the side chain mod-
i®cation in the diphenylmethane series on the binding to
AEBS. Compound 1 is the DPPE and is the lead com-
pound of the series. This compound has a 22-times
weaker anity than tamoxifen for AEBS. Compound 2
contains the dimethylaminoethoxy side chain and is the
diphenylmethane homologue to tamoxifen. Compound 2
has 17-times less anity than tamoxifen for AEBS, and a
slight increase of anity over DPPE. Elimination of N-
alkyl substituant to give the primary amine 7 produced a
370-fold decrease in anity for AEBS compared to
tamoxifen. Replacement of the amine with an unpro-
tonable substituant (3±6) at physiologic pH totally abol-
ished the binding capacity for AEBS. Compound 8, with
a pKa>12, is the most basic compound of this series.
Compound 8 had a very low anity and produced a
612-times decrease of anity compared with tamoxifen.
The loss of anity might be due to the delocalization of
the positive charge and the higher hydration of the
guanidinium group than the tertiary amine found the
side chain of compounds 1, 2, 10±13. The quarter-
narized amonium derivative 9 had a 46-fold decrease in
anity compared with tamoxifen. Compound 10 is a
pyrrolidinic analogue of DPPE. This compound dis-
played a 3-times higher anity than DPPE, showing
that the cyclization of the aliphatic N substituant
increases the anity in the diphenylmethane series.
Replacement of the pyrrolidine ring with the non-pro-
tonable pyrrol ring gave compound 11, which has no
detectable anity for AEBS. The piperidine derivative
12 and the morpholinic derivative 13 have a little higher
anity than DPPE, and a slightly lower anity than the
pyrrolidinyl derivative (10). This illustrates that the ring
part of the amine is important for high anity. Com-
pounds that are essentially non-basic in the Broensted±
Lowry sense have no anity for AEBS. The presence of
a protonable amine is necessary but not sucient for
binding to AEBS as described for the triphenylethylenic
series.7,25,26 As reported for arylbenzofuran, tripheny-
lethylene and other bulky aromatic derivatives, a cyclic
amine such as pyrrolidine, cyclohexylamine and mor-
pholine increased signi®cantly the anity for AEBS.
Interestingly in the diphenylmethane series, the order
aAnalysis for C, H, and N are withinÆ0.4% of the theoretical values.
Ki was determined as described in the caption of Table 1. N.M.=not
measurable.
Table 3. Derivatives of DPPE with altered aromatic moietiesa
Compound
R
X
Mp, ꢀ
C
Formula Ki (nM)
20a
20b
21a
21b
22a
22b
23a
23b
24
c-N(C4H8)O
c-N(C4H8)
c-N(C4H8)O
c-N(C4H8)
c-N(C4H8)O
c-N(C4H8)
c-N(C4H8)O
c-N(C4H8)
C(CH3)2- 188±190 C21H27NO2 48.1Æ2.5
C(CH3)2- 200±202 C21H27NO 10.1Æ1.2
CO-
CO-
118±120 C19H21NO2 102Æ4.5
128±130 C19H21NO 64.5Æ4.2
84±86 C19H23NO3 N.M.
CHOH-
CHOH-
O-
106±108 C19H23NO2 N.M.
170±172 C18H21NO3 2.1Æ0.4
166±168 C18H21NO2 0.8Æ0.2
O-
c-N(C4H8)O
CH=CH- 216±218 C20H23NO2 2010Æ11
CH2-CO- 146±148 C20H23NO3 850Æ12
c-N(C4H8)O CH2-CHOH- 136±138 C20H25NO3 N.M.
25
26
c-N(C4H8)O
aAnalysis for C, H, and N are withinÆ0.4% of the theoretical values. Ki
was determined as described in the caption of Table 1. N.M.=not mea-
surable.
Table 4. ortho-Substituted 1-(2-N-morpholinylethoxy)benzylbenzene
derivativesa
Compound
R
Mp, ꢀC Formula
Ki (nM)
27
28
29
30
31
32
NO2
NH2
N3
111±112 C19H22N2O4 80Æ8.2
82±84* C19H24N2O2 125Æ7.5
122±123 C19H23N4O2 3Æ0.7
212±214 C21H26N2O3 730Æ12.5
NH±CO±CH3
NH±CO±(CH2)2±COOH 150±151 C23H28N2O5 N.M.
NH±CO±(CH2)3±COOH 171±172 C24H30N2O5 N.M.
aAnalysis for C, H, N and O are withinÆ0.4% of the theoretical values.
*Free base. Ki was determined as described in the caption of Table 1.
N.M.=not measurable.

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M. Poirot et al. / Bioorg. Med. Chem. 8 (2000) 2007±2016
of anity is not the same as in other series.7,18,25,26 The
highest anity was obtained with the pyrrolidine deriv-
ative 10 followed by the morpholine derivative 13
with respectively 3.4 and 1.7-times more anity than
DPPE.
anity for AEBS for benzhydrol derivatives (22a,b)
might be due to their lower hydrophobicity and/or the
possible hydration at this position.
trans-Stylbene derivative (24) shows a 114.2 lower an-
ity than its diphenylmethane counterpart. One would
have expected a higher anity for this compound for
AEBS because AEBS does not discriminate between the
cis and trans isomers of tamoxifen.7 It might be that in
the case of compound 24, the conformation of the styl-
bene moiety of the molecule is di€erent from that of the
trans isomer of tamoxifen. The methyl ketone derivative
(25) has 8-times decreased in anity than its benzophe-
nonic counterpart (21a), and a 48-times decrease of a-
nity than its diphenylmethane counterpart. The methyl
methanol derivative (26) have no measurable anity for
AEBS as observed for benzhydrol derivatives (22a) illus-
trating the drastic e€ect on AEBS anity of an hydroxyl
group on the benzylic position. Increasing the distance
between the two phenyl-rings has drastic consequences
on the anity for AEBS. The same e€ect as observed for
one atom X spaced derivatives was produced with the
two atoms X spaced suggesting that the spatial orienta-
tion of the b phenyl ring (Fig. 2) and its distance from the
ammonium side chain might be critical parameters for
anity.
Replacement of the benzyl group in diphenylmethane
backbone
Table 2 shows a series of 1(2-N-morpholinylethoxy)
benzene derivatives used to determine the minimal
structural requirements for binding to AEBS, keeping a
high anity morpholinylethoxy side chain. The analogue
of compound 13 in which the benzyl group has been
omitted (14) or replaced by a methyl group (18) or a
chlorine atom (15) had no detectable anity. When the
benzyl group was replaced with a bulky halogen atom
like bromine (16) and iodine (17) the anity was par-
tially restored but remained low. The tertiobutyl deriva-
tive 19 displayed a moderate anity, which was 8-times
lower than the DPPE. Increasing the bulk of the para
substituent from the side chain on the a phenyl (Fig. 2)
increased the anity for AEBS. This showed that a
bulky hydrophobic group in the para position from the
side chain on the a phenyl is critical for high anity
binding to AEBS.
Modi®cation of X between the two phenyl groups
ortho Substitution from the basic side chain on the
ꢀ phenyl:
Table 3 shows the structure and the anity of various
derivatives in which the aromatic moiety have been
modi®ed by replacing the benzylic methylene with var-
ious X groups. Replacement of the benzylic methylene
with dimethylmethane group gives compounds 20a and
20b that have respectively 1.2 and 6-times higher anity
than DPPE. Compound 20a has a 3-times lower anity
than its diphenylmethane counterparts compound 13,
and compound 20b has the same anity than its diphen-
ylmethane counterpart compound 10. This showed
that cumyl derivatives 20a and 20b have a lower anity
than their diphenylmethane counterparts although the
e€ect was weak. When X is carbonyl group (21a,b) the
anity for AEBS is lowered from 7- and 6-times respec-
tively when compared with their diphenylmethane coun-
terparts. The e€ect of this modi®cation is a decrease of
anity and the order of magnitude is higher for benzo-
phenonic compounds than for cumyl derivatives (20a,b).
When X is a CH2OH- (22a,b) the anity for AEBS is
totally abolished independently of the structure of the
amine side chain. Replacement of X with an oxygen gives
compounds 23a and 23b. These compounds have 8 and
11-times higher anity than their diphenylmethane
homologues, 27 and 70-times higher anity than DPPE
and 1.2 and 3-times higher anity than tamoxifen. The
highest anity was obtained with the less sterically hin-
dered sp3 atom, suggesting that the angle between the
two planes de®ned by the benzene rings and their spatial
positioning might be important. Among the compounds
that have measurable anities, pyrrolidinic compounds
have higher anity for AEBS than their morpholinic
counterparts; however the order of magnitude in the
change of anity is not the same for a given X accord-
ing to the structure of the amine side chain. The loss of
The presence of various groups in the ortho position on
the a benzene ring from the side chain (Fig. 2) and its
repercussion on the anity for AEBS is shown in Table 4.
These modi®cations were originally carried out with a
view of developing compounds useful for anity label-
ing27,28 or anity chromatography of AEBS. The pre-
sence of a nitro group (27) induced a 5-times decrease in
anity, and a 7-times decrease was observed with the
amino group (28). Transformation into an azido group
(29) produced an increase of anity compared with
compound 13. Amidation of the compound 28 give the
compound 30, which had a 41-fold lower anity than
tamoxifen. Succinylation and glutamylation abolished the
anity for AEBS. Aromatic substitution might a€ect
electronic density, which is important for stacking inter-
actions, but other parameters such as steric hindrance,
hydrophilicity and subsequently hydration might have
occurred. Nitro- and amino-groups are opposite in term
of electronic e€ect, but induced anity decreases of the
same order of magnitude, suggesting that this e€ect
might be due to hydration. For compounds 31 and 32,
intramolecular electrostatic interaction with the amine
side-chain might be involved in the loss of anity.
Molecular modeling
The three-dimensional structure of tamoxifen has been
determined.29 A stick model constructed from the
atomic coordinates is shown in Figure 3. Tamoxifen
exhibits a propeller structure which is characteristic of
triphenylethylenic compounds. The amine-aryl ether
chain of tamoxifen is extended as in the calculated mini-
mal energy conformation of the DPPE. However unlike

M. Poirot et al. / Bioorg. Med. Chem. 8 (2000) 2007±2016
2011
Figure 3. Molecular structure of tamoxifen and DPPE. Superimposition and intersection of their van der Waals volumes. On the left: three-
dimensional structure of tamoxifen (A) depicted using coordinates from the X-ray crystallographic structure analysis by Precigoux et al.29 On the
right: minimum energy conformation of DPPE (B). On the center: superimposition and intersection of the van der Waals volume (A+B) of
tamoxifen and DPPE.
the DPPE, the aromatic part of tamoxifen is not ¯exible:
phenyl groups are conformationally restricted with a
high-energy barrier of rotation. The van der Waals
volume of tamoxifen is 321.95 A3. In its calculated
minimum energy conformation, DPPE has a van der
Waals volume of 207.63 A3. We superimposed the van der
Waals volumes of the minimum energy conformation of
the DPPE and the van der Waals volumes of the crys-
tallographic structure of tamoxifen by superimposition
of the following atoms: N±CH2±CH2±O±C6H4 (Fig. 3)
as recently reported by our group for PBPE and tamox-
ifen.10 The intersection of the van der Waals volume of
DPPE and tamoxifen matches with the diphenylmethane
and N,N-dimethylaminoethoxy moieties of tamoxifen.
80% of the volume of DPPE matches with the diphen-
ylmethane and the basic side chain of the tamoxifen
(Fig. 3). There is an excellent superimposition although
the carbon bearing the phenyl û is sp3 in diphenylmeth-
ane and sp2 in tamoxifen. Phenyl propene of tamoxifen,
which is linked to a sp2 carbon, introduces constraints
that reduce the angle de®ned by the benzyllic carbon and
the bonded phenyl rings.29 In Table 5 we reported the
van der Waals volume of each compound that binds
AEBS as calculated when they are in their minimal
Table 5. Van der Waals volume intersection between new AEBS
ligands and tamoxifen
Compound
Van der Waals
volume in A3
Volume intersection
in A3
Ki
(nM)
Tamoxifen
1
2
7
8
321.95
250.75
221.64
192.21
222.94
242.30
256.66
248.99
189.43
199.83
224.26
279.61
271.86
248.35
241.38
258.65
265.71
267.46
259.51
290.25
2.5
55.6
42.3
270
1530
8.8
28.1
17.6
3820
1630
248
48.1
10.1
102
207.63
209.49
177.26
144.12
201.63
199.56
199.10
146.75
153.31
167.32
180.44
206.12
174.72
177.58
154.67
165.29
180.12
175.80
153.27
10
12
13
16
17
19
20a
20b
21a
21b
24
25
27
28
30
64.5
2010
850
80
125
Figure 4. Relationship between van der Waals volume intersection
and pKi of the compounds with AEBS binding, a linear correlation
(r=0.84, p<0.0001, n=18) is drawn.
730

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M. Poirot et al. / Bioorg. Med. Chem. 8 (2000) 2007±2016
energy conformation. We have reported the values of
the intersection of their van der Waals volumes and those
of tamoxifen in its crystallographic state. When the anity
for AEBS (pki) is plotted against the intersection of the
van der Waals volume we obtain a linear correlation
(Fig. 4) (r=0.84, P<0.0001, n=18). This delineates for
the ®rst time that a linear quantitative structure-anity
relationship can be established between these two para-
meters. This illustrates that the rigid triphenylethylenic
moiety of tamoxifen de®nes a spatial relationship driving
the occupancy of the tamoxifen within AEBS and the
binding of compounds from our series.
with UV light (254 nM) or iodine. Melting points were
uncorrected and measured on a Ko¯er apparatus.
Molecular structure analysis
Computational chemical calculations were performed
on a Silicon Graphics Indigo workstation using insight
ii version 97.0 (Biosim/MSI). Minimum energy con-
formations were calculated using the Discover module
(2.9.7/95.0/3.0.0) with CVFF force®eld. Van der Waals
volumes and van der Waals volume intersections were
determined using the Search-Compare module version
97.0 (Biosim/MSI).
Compounds 23a and 23b, however, do not enter in this
correlation. The spatial positioning of phenyl û of these
compounds in their minimal energy conformation does
not match with the phenyl û of tamoxifen and might
de®ne a more tightly interacting area in the binding site.
Structure±anity studies will be investigated further on
the phenoxyphenol derivatives.
Ligand binding assay (AEBS)
Anti-estrogen binding sites were labeled as previously
described using rat liver membranes,28 a rich source of
AEBS, and [3H]tamoxifen in the presence of 1mM of 17û-
estradiol to mask residual estrogen receptors.28 Assays
were performed in 50 mM Tris±HCl, 1 mM EDTA,
12 mM thioglycerol at pH 7.4 for 18 h at 4 ꢀC in a volume
of 200 mL with 80 mg of microsomal proteins and 3 nM of
[3H]tamoxifen and 12 concentrations of unlabelled test
ligand ranging from 0.1 nM to 10 000 nM. Assays with
[3H]tamoxifen included 1 mM of 17û-estradiol. IC50
values were determined using the iterative curve ®tting
program GraphPad prism. IC50 values were converted
into the apparent Ki using the Cheng±Pruso€ equation,30
and the Kd values. Non-speci®c binding was carried out
with 2 mM of tamoxifen. Assays were terminated by
loading 150 mL of the incubate on a 3.5 mL Sephadex
LH-20 column equilibrated with 50mM Tris±HCl, 1 mM
EDTA, 12mM thioglycerol at pH 7.4. Elutions were
carried out by adding 1 mL fractions of 50 mM Tris±
HCl, 1 mM EDTA, and 12 mM thioglycerol at pH 7.4.
The eluates were collected and counted in ready safe
scintillant (Beckman).
Conclusion
We have described the synthesis and AEBS binding a-
nities of a series of diphenyl methane based tamoxifen
derivatives that are selective for AEBS over the estrogen
receptor. None of the compounds that we prepared had
detectable anity for the estrogen receptor. Cyclic
amines on the side chain, hydrophobic groups in the para
position from this side chain on the phenyl ring, the spa-
tial orientation and the distance between the two phenyl
rings are critical for the anity to AEBS. Three-dimen-
sional analysis shows that a linear correlation can be
established between the intersection of the van der Waals
volume of tamoxifen and those of the new compounds.
We have synthesized compounds with higher anity for
AEBS than DPPE, the lead compound from the diphen-
ylmethane series, and two of them have higher anity
than tamoxifen. These compounds will help to character-
ize further AEBS-driven cytotoxicity and potentiation
of the chemotherapeutic index of cytotoxic drugs.
Ligand binding assay (estrogen receptor)
ER binding experiments were conducted using estrogen
receptors extracted from MCF-7 cells as previously
described.31 Brie¯y MCF-7 (from ATCC) were grown
to 80% con¯uency in DMEM medium (GiBCO BRL)
supplemented with 10% FCS. Cells were then scraped,
and washed twice with PBS. After centrifugation for
10 min at 1500 rpm, the cells were resuspended in TM
bu€er (20 mM Tris, HCl pH 7.4; sodium molybdate,
20 mM). Cells were broken by freeze-thaw lysis of the
cell pellets in an equal volume of TM bu€er. Cytosolic
receptors were prepared by a 105 000 gÂ60 min cen-
trifugation at 0 ꢀC, and then stored at 80 ꢀC. This
cytosolic receptor solution was diluted to 60% in TM
bu€er, and then incubated with the corresponding
ligand 18 h at 4 ꢀC in a volume of 100 mL with 50 mg of
protein and 2 nM of [3H]estradiol and two concentra-
tions (1 and 10 mM) of unlabeled test ligand. Assays
were terminated by loading 65 mL of the incubate on a
1.2 mL SephadexTM LH-20 column equilibrated with
the TM bu€er. The ¯ow through was collected and
counted for radioactivity in ready safe scintillant
(Beckman).
Experimental
Materials and methods
1H NMR were recorded at 200 MHz on a Brucker
AC200 spectrometer. Chemical shifts are given in parts
per million and tetramethylsilane was used as the internal
standard for spectra obtained in DMSO-d6 or CDCl3.
All J values are given in Hz. Mass spectra were recorded
on a variant MAT 311 A mass spectrometer. Infrared
spectra were recorded on a Perkin-Elmer 599 spectro-
photometer. Elemental analysis was carried out by the
microanalytical service laboratory of the Ecole Superieure
de Chimie of the Universite Paul Sabatier, France.
Reagents and solvents were used as obtained from com-
mercial suppliers without further puri®cation. Column
chromatography was performed using silica gel, and
reaction progress was determined by TLC analysis on
silica gel coated aluminum plates. Visualization was

M. Poirot et al. / Bioorg. Med. Chem. 8 (2000) 2007±2016
2013
Chemical synthesis
containing 1.1 equivalents of CBr4 (1.3 g, 13.1 mmoles)
at 0 ꢀC. 1.2 equivalents of triphenylphosphine were then
added. The mixture was then stirred for 1 h at 0 ꢀC and
then 1 h at room temperature. The solution was extracted
3 times with water and then dried over MgSO4. Eva-
poration of CH2Cl2 and recrystalization in a minimum
of methyl alcohol gave 2.05 g (69%) of a white solid: mp
60±62 ꢀC; 1H NMR d (DMSO-d6) 7.1±7.6 (m, 9H, ArH),
4.28 (t, 2H, ArOCH2), 3.91 (s, 2H, ArCH2), 3.62 (t, 2H,
CH2Br), MS m/z 291 (M+).
[2-(4-Benzyl-phenoxy)-ethyl]-diethyl-amine hydrochloride
(DPPE) (1). 2-Diethylaminoethylchloride (0.50 g, 2.72
mmoles) was added to a solution of 4-benzylphenol
(0.58 g, 2.72 mmoles) and K2CO3 (0.69 g, 5.44 mmoles)
in 15 mL of a mixture of dry DMF:acetone (1:1). The
reaction mixture was maintained for 18 h at 60 ꢀC. The
mixture was then ®ltered and poured into 100 mL of
cold water and extracted twice with 100 mL of diethyl
ether. The organic layer was washed 3 times with 10mL of
an aqueous solution of sodium hydroxide (0.1N) and then
3 times with 10mL of brine. The etherous organic layer was
then extracted with 5 mL of 12N hydrochloric acid. The
aqueous layer was collected and evaporated. The white
solid was then recrystalized in a mixture of isopropanol:
acetone (3:1) to give 0.78g (90%) of white crystals as the
2-[2-(4-Benzyl-phenoxy)-ethyl]-isoindole-1, 3-dione (6).
0.5 g of compound 5 (1.7 mmoles) and 1.1 equivalents of
potassium phtalimide in 15 mL of dry DMF were mixed
at 60 ꢀC for 12 h. The mixture was poured into 100 mL
of water and extracted twice with 25mL of CH2Cl2. The
organic layer was washed three times with 15mL of
NaOH 0.1N, then three times with 15mL of brine. The
organic layer was dried over MgSO4 before being evapo-
rated, and gave 0.58g (95%) of a white solid, which was
recrystalized in hexane. Mp 80±82 ꢀC; 1H NMR d
(CDCl3) 7.1±8.2 (m, 13H, ArH), 3.86 (s, 2H, ArCH2),
4.22 (t, J=4.6 Hz, 2H, CH2N), 4.05 (t, J=4.6 Hz, 2H,
ArOCH2); MS m/z 357 (M+).
hydrochloride salt: mp 156±158 ꢀC; H NMR d (DMSO-
d6) 7.1±7.6 (m, 9H, ArH), 3.91 (s, 2H, ArCH2), 4.4 (t,
J=5.8 Hz, 2H, ArOCH2), 2.82 (t, J= 5.8 Hz, 2H, CH2N),
2.63 (q, 4H, NCH2), 1.2 (t, 6H, CH3). MS m/z 283 (M+1).
1
[2-(4-Benzyl-phenoxy)-ethyl]-dimethyl-amine hydrochloride
(2). The same procedure was used as for compound 1
using the corresponding chloroethylamine. The white
solid was then recrystalized in a mixture of isopropanol:
acetone (3:1) to give 0.76g (2.4 mmol, 88%) of white crys-
tals as the hydrochloride salt: mp 178±180 ꢀC, 1H NMR d
(DMSO-d6) 7.1±7.6 (m, 9H, ArH), 3.91 (s, 2H, ArCH2),
4.4 (t, J=5.8Hz, 2H, ArOCH2), 2.82 (t, J=5.8Hz, 2H,
CH2N), 2.62 (s, 6H, NCH3). MS m/z 256 (M+1).
2-(4-Benzyl-phenoxy)-ethylamine hydrochloride (7). A
solution of 0.5 g of compound 6 (1.4 mmoles) and 5
equivalents of hydrazine hydrate in 10 mL of ethanol
was heated to re¯ux for 3.5 h and then cooled. Removal
of the solvent gave a yellow oil which was extracted with
diethylether, washed 3 times with 10 mL of NaOH 0.1N,
then three times with 10 mL of brine and ®nally was
extracted with 5 mL of HCl 12N. The extract was eva-
porated and the recrystalized in isopropanol to give 0.3 g
of white crystals as the hydrochloride salt: yield 81%; mp
226±228 ꢀC; 1H NMR d (DMSO-d6) 7.1±7.6 (m, 9H,
ArH), 3.87 (s, 2H, ArCH2), 3.82 (t, 2H, ArOCH2), 2.85
(t, 2H, CH2N), 2.15 (s, 2H, NH2, exchangeable with
D2O); MS m/z 228 (M+1).
2-(4-Benzyl-phenoxy)-ethanol (3). The same procedure
was used as for compound 1 with 2-chloro1ethanol
instead of chloroethylamine. The reaction mixture was
maintained for 18h at 60 ꢀC. The mixture was then ®ltered,
poured into 100 mL of cold water and extracted twice
with 100 mL of diethyl ether. The organic layer was
washed 3 times with 10 mL of an aqueous solution of
sodium hydroxide (0.1N) and then 3 times with 10 mL
of brine. The ®nal etheral solution was evaporated
under reduced pressure and the white solid was then
N-[2-(4-Benzyl-phenoxy)-ethyl]-guanidine (8). A solution
of 0.3 g (1.13 mmoles) of compound 7 and 3.5-dim-
ethylpyrazole-1-carboxamidine nitrate in 5 mL of ethanol
was heated to re¯ux for 4 h and then cooled. Removal
of the solvent in vacuo and recrystalization of the resi-
due from hexane gave the product 8 (350 mg, 93%). Mp
102±104 ꢀC; 1H NMR d (DMSO-d6) 7.1±7.6 (m, 9H,
ArH), 3.86 (s, 2H, ArCH2), 4.05 (t, J=4.6 Hz, 2H,
CH2N), 3.12 (t, J=4.6 Hz, 2H, ArOCH2); MS m/z 270
(M+1).
recrystalized in hexane. Yield 85%; mp 70±72 ꢀC; H
1
NMR d (DMSO-d6) 7.1±7.6 (m, 9H, ArH), 4.01 (m, 4H,
OCH2CH2O), 3.90 (s, 2H, ArCH2); MS m/z 228 (M+).
Methanesulfonic acid 2-(4-benzyl-phenoxy)-ethyl ester (4).
2 g (8.8 mmoles) of compound 3 was dissolved in 20 mL
of CH2Cl2 containing a 50% excess of triethylamine
(1.3 g, 13.1 mmoles) at 0 ꢀC. 1.1 equivalents of methane-
sulfonylchloride was added dropwise over 10 min. The
mixture was stirred for 15 min before being worked up 3
times with 10 mL of a cold 10% solution of HCl, then 3
times with 10 mL of a saturated solution of NaHCO3,
and ®nally 3 times with 10 mL of brine. The organic layer
was then dried over MgSO4 and evaporated to give 2.4 g
of a white solid. Yield 89%, mp 60±62 ꢀC, NMR d
(DMSO-d6) 7.1±7.6 (m, 9H, ArH), 4.20 (t, 2H, ArOCH2),
4.05 (t, 2H, CH2OSO2), 3.90 (s, 2H, ArCH2), 3.69 (s, 3H,
SO2CH3). MS m/z 306 (M+).
[2-(4-Benzyl-phenoxy)-ethyl]-trimethyl-ammonium (9).
0.5 g of compound 2 was added in its free base state to
an excess of methyl iodide in 10 mL of diethylether. The
mixture was maintained under constant stirring over-
night at room temperature. A precipitate appeared
which was collected by ®ltration and washed extensively
with diethylether. The white precipitate was recrystal-
ized in isopropyl alcohol to give 0.55 g (82%) of com-
1
pound 9. Mp 138±140; H NMR d (DMSO-d6) 7.1±8.2
(m, 9H, ArH), 3.92 (s, 2H, ArCH2), 3.35 (t, J=4.6 Hz,
2H, CH2N), 4.05 (t, J=4.6 Hz, 2H, ArOCH2), 3.2 (s,
9H, NCH3); MS m/z 358 (M+).
2-(4-Benzyl-phenoxy)-ethylbromine (5). 2 g (8.8 mmoles)
of compound 3 was dissolved in 20 mL of CH2Cl2

2014
M. Poirot et al. / Bioorg. Med. Chem. 8 (2000) 2007±2016
1-[2-(4-Benzyl-phenoxy)-ethyl]-1H-pyrrole (10). A solu-
tion 0.5 g of compound 5 (1.7 mmoles) and 1.1 equivalents
of pyrrole in 15 mL of dry DMF were mixed at 60 ꢀC for
12 h. The mixture was poured into 100 mL of water and
extracted twice with 25 mL of CH2Cl2. The organic
layer was washed three times with 15 mL of NaOH
0.1N, then three times with 15 mL of brine. The organic
layer was dried over MgSO4 before being evaporated and
gave 0.58 g (95%) of a yellow liquid. 1H NMR d
(DMSO-d6) 6.6±7.6 (m, 11H, ArH+pyrrol a-H), 6.2 (m,
2H pyrrol û-H), 3.91 (s, 2H, ArCH2), 4.06 (t, J=5.8 Hz,
2H, ArOCH2), 4.18 (t, J=5.8 Hz, 2H, CH2N); MS m/z
278 (M+).
adjacent to O), 2.30 (t, 4H, CH2 ring adjacent to N); MS
m/z 334 (M+1).
4-(2-p-Tolyloxy-ethyl)-morpholine hydrochloride (18).
Yield 80%; mp 208±210 ꢀC; 1H NMR d (DMSO-d6)
6.7±6.9 (m, 4H, ArH), 4.01 (t, J=5.8 Hz, 2H, ArOCH2),
2,82 (t, J=5.8 Hz, 2H, CH2N), 3.65 (t, 4H, CH2 ring
adjacent to O), 2.35 (s, 3H, ArCH3), 2.30 (t, 4H, CH2
ring adjacent to N); MS m/z 223 (M+1).
4-[2-(4-tert-Butyl-phenoxy)-ethyl]-morpholine hydrochlo-
ride (19). Yield 78%; mp 190±192 ꢀC; 1H NMR d
(DMSO-d6) 6.7±7.1 (m, 4H, ArH), 4.01 (t, J=5.8 Hz, 2H,
ArOCH2), 2,82 (t, J=5.8 Hz, 2H, CH2N), 3.65 (t, 4H,
CH2 ring adjacent to O), 1.32 (s, 9H, CCH3), 2.30 (t,
4H, CH2 ring adjacent to N); MS m/z 264 (M+1).
Compounds 11±21b were prepared according to the
method described above for compound 1.
1-[2-(4-Benzyl-phenoxy)-ethylꢀ]-pyrrolidine hydrochloride
(11). Yield 72%; mp 170±172 C; 1H NMR d (DMSO-d6)
7.1±7.6 (m, 9H, ArH), 3.85 (s, 2H, ArCH2), 4.05 (t,
J=5.8Hz, 2H, ArOCH2), 2.68 (t, J=5.8 Hz, 2H, CH2N),
2.52 (m, 4H, ring NCH2), 1.7 (m, 4H ring CH2 non-
adjacent to N). MS m/z 282 (M+1).
4-{2-[4-(1-Methyl-1-phenyl-ethyl)-phenoxy]-ethyl}-mor-
pholine hydrochloride (20a). Yield 83%; mp 188±190 ꢀC;
1H NMR d (DMSO-d6) 6.89±7.28 (m, 9H, ArH), 4.39±
4.44 (t, J=5.8 Hz, 2H, ArOCH2), 3.22 (t, J=5.8 Hz,
2H, CH2N), 3.85±3.91 (t, 4H, CH2 ring adjacent to O),
3.43±3.51 (t, 4H, CH2 ring adjacent to N), 1.63 (s, 6H,
CCH3); MS m/z 326 (M+1).
1-[2-(4-Benzyl-phenoxy)-ethyl]-piperidine hydrochloride
ꢀ
1
(12). Yield 75%; mp 180±182 C; H NMR d (DMSO-
d6) 7.1±7.6 (m, 9H, ArH), 3.96 (s, 2H, ArCH2), 4.5 (t,
J=5.8Hz, 2H, ArOCH2), 2.51 (t, J=5.8 Hz, 2H, CH2N),
2.25 (t, 4H, CH2 ring adjacent to N), 1.73 (m, 6H ring
non adjacent to N); MS m/z 296 (M+1).
1-{2-[4-(1-Methyl-1-phenyl-ethyl)-phenoxy]-ethyl}-pyrro-
lidine hydrochloride (20b). Yield 85%; mp 200±202 ꢀC;
1H NMR d (DMSO-d6) 6.89±7.31 (m, 9H, ArH), 4.29±
4.34 (t, J=5.8 Hz, 2H, ArOCH2), 3.08±3.11 (t, J=
5.8 Hz, 2H, CH2N), 3.53±3.57 (m, 4H, ring NCH2),
1.89±1.97 (t, 4H, CH2 ring non adjacent to N), 1.62 (s,
6H, CCH3); MS m/z 309 (M+1).
4-[2-(4-Benzyl-phenoxy)-ethyl]-morpholine hydrochloride
ꢀ
1
(13). Yield 78%; mp 185±186 C; H NMR d (DMSO-
d6) 7.1±7.6 (m, 9H, ArH), 3.96 (s, 2H, ArCH2), 4.2 (t,
J=5.8Hz, 2H, ArOCH2), 2.69 (t, J=5.8 Hz, 2H, CH2N),
3.95 (t, 4H, CH2 ring adjacent to O), 2.25 (t, 4H, CH2
ring adjacent to N); MS m/z 298 (M+1).
[4-(2-Morpholin-4-yl-ethoxy)-phenyl]-phenyl-methanone
hydrochloride (21a). Yield 70%; mp 118±120 ꢀC; 1H
NMR d (DMSO-d6) 6.9±7.71 (m, 9H, ArH), 4.02 (t,
J=5.8Hz, 2H, ArOCH2), 2,80 (t, J=5.8 Hz, 2H, CH2N),
3.67 (t, 4H, CH2 ring adjacent to O), 2.38 (t, 4H, CH2 ring
adjacent to N); MS m/z 312 (M+1).
4 - (2 - Phenoxy - ethyl) - morpholine hydrochloride (14).
Yield 75%; mp 189±190 ꢀC; 1H NMR d (DMSO-d6)
6.8±7.2 (m, 5H, ArH), 4.01 (t, J=5.8 Hz, 2H, ArOCH2),
2,82 (t, J=5.8 Hz, 2H, CH2N), 3.65 (t, 4H, CH2 ring
adjacent to O), 2.30 (t, 4H, CH2 ring adjacent to N); MS
m/z 208 (M+1).
Phenyl-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-methanone
hydrochloride (21b). Yield 75%; mp 128±130 ꢀC; 1H
NMR d (DMSO-d6) 6.9±7.71 (m, 9H, ArH), 4.05 (t,
J=5.8Hz, 2H, ArOCH2), 2,68 (t, J=5.8 Hz, 2H, CH2N),
2.28 (m, 4H, ring NCH2), 1.6 (t, 4H, CH2 ring non adja-
cent to N); MS m/z 296 (M+1).
4-[2-(4-Chloro-phenoxy)-ethyl]-morpholine hydrochloride
(15). Yield 83%; mp 222±224 ꢀC; 1H NMR d (DMSO-d6)
6.8±7.2 (m, 4H, ArH), 4.01 (t, J=5.8 Hz, 2H, ArOCH2),
2,82 (t, J=5.8Hz, 2H, CH2N), 3.65 (t, 4H, CH2 ring
adjacent to O), 2.30 (t, 4H, CH2 ring adjacent to N). MS
m/z 242 (M+1).
[4-(2-Morpholin-4-yl-ethoxy)-phenyl]-phenyl-carbinol
hydrochloride (22a). To a mixture of 100 mg of com-
pound 21a (0.32 mmol) in 2.5 mL tri¯uoroacetic acid
and 2.5 mL of CH2Cl2 was added 0.33 mmol of NaBH4
at 0 ꢀC The mixture was stirred at room temperature
during 30 min until the starting material disappeared.
The residue was hydrolyzed on ice, and the acid was
neutralized with a solution of NaHCO3. The aqueous
phase was then extracted with 10 mL diethylether and
the extract was washed 3 times with 5 mL of brine. The
organic layer was dried over MgSO4 and mixed with an
etheral solution of 1N HCl, which produced the pre-
cipitation of compound 22a. Yield 48%; mp 84±86 ꢀC;
1H NMR d (DMSO-d6) 7.1±7.6 (m, 9H, ArH), 5.8 (s,
1H, CHOH), 4.02 (t, J=5.8 Hz, 2H, ArOCH2), 2,80 (t,
J=5.8 Hz, 2H, CH2N), 3.67 (t, 4H, CH2 ring adjacent
4-[2-(4-Bromo-phenoxy)-ethyl]-morpholine hydrochloride
(16). Yield 82%; mp 180±182 ꢀC; NMR d (DMSO-d6)
6.8±7.3 (m, 4H, ArH), 4.01 (t, J=5.8 Hz, 2H, ArOCH2),
2,82 (t, J=5.8 Hz, 2H, CH2N), 3.65 (t, 4H, CH2 ring
adjacent to O), 2.30 (t, 4H, CH2 ring adjacent to N); MS
m/z 287 (M+1).
4-[2-(4-Iodo-phenoxy)-ethyl]-morpholine hydrochloride
(17). Yield 82%; mp 178±180ꢀC; 1H NMR d (DMSO-d6)
6.6±7.5 (m, 4H, ArH), 4.01 (t, J=5.8Hz, 2H, ArOCH2),
2,82 (t, J=5.8 Hz, 2H, CH2N), 3.65 (t, 4H, CH2 ring

M. Poirot et al. / Bioorg. Med. Chem. 8 (2000) 2007±2016
2015
to O), 2.38 (t, 4H, CH2 ring adjacent to N); MS m/z 314
(M+1).
3.67 (t, 4H, CH2 ring adjacent to O), 2.38 (t, 4H, CH2
ring adjacent to N), 2.02 (s, 3H, NHCO-CH3); MS m/z
355 (M+1).
[4-(2-Pyrrolidin-4-yl-ethoxy)-phenyl]-phenyl-methanol
hydrochloride (22b). This compound was prepared
according to the method described above for compound
22a. Yield 52%; mp 106±108 ꢀC; 1H NMR d (DMSO-d6)
7.1±7.6 (m, 9H, ArH), 5.8 (s, 1H, CHOH), 4.05 (t,
J=5.8Hz, 2H, ArOCH2), 2,68 (t, J=5.8 Hz, 2H, CH2N),
2.28 (m, 4H, ring NCH2), 1.6 (t, 4H, CH2 ring non-
adjacent to N); MS m/z 298 (M+1).
N-[5-Benzyl-2-(2-morpholin-4-yl-ethoxy)-phenyl]-succi-
namic acid (31). Succinic anhydride (1 mmole) was added
to a solution of 28 (200mg, 0.64mmole) in 5 mL of diox-
ane 1±4 for 1 h with stirring at room temperature. The
white precipitate was collected, ®ltered and washed with
diethylether to give 250 mg of compound 33. Yield 95%,
mp 150±151 ꢀC; H NMR d (DMSO-d6) 7.1±7.7 (m, 8H,
1
ArH), 4.02 (t, J=5.8Hz, 2H, ArOCH2), 3.91 (s, 2H,
ArCH2), 2,80 (t, J=5.8Hz, 2H, CH2N), 3.67 (t, 4H, CH2
ring adjacent to O), 2.38 (t, 4H, CH2 ring adjacent to N),
2.60±2.70 (m, 4H, NHCOCH2CH2COOH); MS m/z 412
(M+1).
Compounds 23a±25 were prepared according to the
method described above for compound 1.
1-[2-(4-Phenoxy-phenoxy)-ethyl]-morpholine hydrochlo-
ride (23a). Yield 82%; mp 170±172 ꢀC; 1H NMR d
(DMSO-d6) 6.75±7.22 (m, 9H, ArH), 4.02 (t, J=5.8 Hz,
2H, ArOCH2), 2,80 (t, J=5.8 Hz, 2H, CH2N), 3.67 (t,
4H, CH2 ring adjacent to O), 2.38(t, 4H, CH2 ring
adjacent to N); MS m/z 300 (M+1).
4-[5-Benzyl-2-(2-morpholin-4-yl-ethoxy)-phenylcarbamoyl]-
butyric acid (32). The same procedure as for compound
31 was used. Yield 98%; mp 171±172 ꢀC; H NMR d
1
(DMSO-d6) 7.1±7.7 (m, 8H, ArH), 4.02 (t, J=5.8 Hz,
2H, ArOCH2), 3.91 (s, 2H, ArCH2), 2,80 (t, J=5.8 Hz,
2H, CH2N), 3.67 (t, 4H, CH2 ring adjacent to O), 2.38
(t, 4H, CH2 ring adjacent to N), 1.95±2.22 (m, 6H,
NHCOCH2CH2CH2COOH); MS m/z 426 (M+1).
1-[2-(4-Phenoxy-phenoxy)-ethyl]-pyrrolidine hydrochlo-
ride (23b). Yield 78%; mp 166±168 ꢀC; 1H NMR d
(DMSO-d6) 6.75±7.22 (m, 9H, ArH), 4.05 (t, J=5.8 Hz,
2H, ArOCH2), 2,68 (t, J=5.8 Hz, 2H, CH2N), 2.28 (m,
4H, ring NCH2), 1.6 (t, 4H, CH2 ring non adjacent to
N); MS m/z 284 (M+1).
Acknowledgements
4-[2-(4-Styryl-phenoxy)-ethyl]-morpholine hydrochloride
This paper is dedicated to the memory of Dr Moussa
Traore. This work was supported in part by grants from
the Institut National de la Sante et de la Recherche
Medicale, the Conseil Regional de la Haute-Garonne,
the Association pour la Recherche contre le Cancer, the
Ligue Nationale contre le Cancer (delegation regionale
de la Haute Garonne). Philippe De Medina was sup-
ported by a predoctoral followship from the Ministere
de la Recherche et de la Technologie.
(24). Yield 83%; mp 216±218 ꢀC; H NMR d (DMSO-
1
d6) 6.75±7.45 (m, 11H, ArH), 4.02 (t, J=5.8 Hz, 2H,
ArOCH2), 2,80 (t, J=5.8 Hz, 2H, CH2N), 3.67 (t, 4H,
CH2 ring adjacent to O), 2.38 (t, 4H, CH2 ring adjacent
to N); MS m/z 310 (M+1).
1-[4-(2-Morpholin-4-yl-ethoxy)-phenyl]-2-phenyl-ethanone
hydrochloride (25). Yield 82%; mp 146±148 ꢀC; 1H
NMR d (DMSO-d6) 6.89±7.77 (m, 9H, ArH), 4.02 (t,
J=5.8 Hz, 2H, ArOCH2), 3.81 (s, 2H, ArCH2), 2,80 (t,
J=5.8 Hz, 2H, CH2N), 3.67 (t, 4H, CH2 ring adjacent
to O), 2.38 (t, 4H, CH2 ring adjacent to N); MS m/z 326
(M+1).
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