Journal of Steroid Biochemistry and Molecular Biology 70 (1999) 59±71

www.elsevier.com/locate/jsbmb

MR 20492 and MR 20494: two indolizinone derivatives that

strongly inhibit human aromatase
P. Auvray a, P. Sourdaine a, S. Moslemi a, G.-E. SeÂralini a,*, P. Sonnet b, C. Enguehard b,
J. Guillon b, P. Dallemagne b, R. Bureau b, S. Rault b
a
IBBA, Laboratoire de Biochimie et Biologie MoleÂculaire, Universite de Caen, Esplanade de la Paix, 14032 Caen cedex, France
b
C.E.R.M.N., Laboratoire de Pharmacochimie, 1 rue VaubeÂnard, 14032 Caen cedex, France
Received and accepted 26 February 1999

Abstract

In this study, we describe the synthesis of a new family of indolizinone derivatives designed to ®t an extrahydrophobic pocket
within the active site of aromatase and to strongly inhibit human aromatase. This could help improve the speci®city of the
inhibitors. Equine aromatase, very well characterized biochemically, is used as a comparative model. Indeed, in a previous
comparison between both human and equine aromatases, we described the importance of the interaction between the inhibitor
and this pocket for the indane derivative MR 20814. MR 20492 and MR 20494 are more potent inhibitors of human aromatase
(Ki/Km: 1.0 2 0.3 and 0.52 0.3, respectively). The Ki/Km for MR 20494 is slightly higher than that obtained for fadrozole
(0.1 20.0) and Ki/Km for both indolizinone derivatives are lower than those obtained for 4-hydroxyandrostenedione (1.92 0.8)
and MR 20814 (8.12 .7). These new compounds are not enzyme inactivators. Moreover, as indicated by the higher Ki/Km values
obtained with equine enzyme (9.0 2 0.6 and 6.1 2 1.6 for MR 20492 and MR 20494, respectively), both human and equine
aromatase active sites appear to be structurally di€erent. Di€erence absorption spectra study (350±500 nm) revealed that
MR20492 and MR20494 were characterized by a combination of type-I and -II spectra with both enzymes. This result could be
due to the isomerization of the molecule in polar solvent (Z and E forms). The evaluation of these new molecules, as well as 4-
hydroxyandrostenedione and fadrozole, on aromatase activity in transfected 293 cell cultures evidenced a strong inhibition (IC50:
0.20 2 0.03 mM, 0.20 2 0.02 mM and 0.50 2 0.40 mM for MR 20494, fadrozole and 4-OHA, respectively) except for MR 20492
(3.9 20.9 mM) and MR 20814 (10.5 2 0.6 mM). These results proved that these molecules formed part of a promising family of
potent inhibitors and that they penetrate 293 cells, without evidencing any cytotoxicity in Hela cells with MTT assay. This is
thus encouraging for the development of new drugs for the treatment of estrogen-dependent cancers, these molecules also
constitute new tools for understanding the aromatase active site. # 1999 Elsevier Science Ltd. All rights reserved.

1. Introduction inhibition constitutes an approach for studying the

roles of estrogen biosynthesis in various physiological
Aromatase is the enzymatic complex responsible for or pathological processes, such as estrogen-dependent
estrogen biosynthesis, formed by the speci®c cyto- cancers. To avoid inhibition of other enzymes, the
chrome P450 aromatase (aromatase herein) and an selectivity of the inhibitors is crucial. However, nonse-
ubiquitous ¯avoprotein NADPH cytochrome P450 re- lective aromatase inhibitors, like nonsteroidal amino-
ductase. The study of structure±activity relationships glutethimide [1,2], which is a well known and long-
of aromatase inhibitors is very helpful for evaluating used therapeutic agent, could a€ect enzymes control-
the active site structure of the enzyme. Moreover, its ling the production of other steroids and induce signi®-
cant side e€ects. Recently, many new potent and
selective inhibitors have been designed and are now
* Corresponding author. Tel.: +33-2-3156-5489; fax: +33-2-3156- under clinical development. Some of these are: (i) 4-
5320. hydroxyandrostenedione (4-OHA) or formestane, a

0960-0760/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 6 0 - 0 7 6 0 ( 9 9 ) 0 0 0 9 3 - X
60 P. Auvray et al. / Journal of Steroid Biochemistry and Molecular Biology 70 (1999) 59±71

Scheme 1. Synthesis of compounds 1-5.

very speci®c steroidal inhibitor used in therapy [3], (ii) indane derivatives (MR 20814: 3-amino-5-methoxy-2-
fadrozole or CGS 16949A, highly potent in vitro [4], (pyridin-4-ylmethyl)inden-1-one and MR 20818: 3-
(iii) vorozole, highly speci®c for aromatase [5,6], (iv) amino-2-(pyridin-4-ylmethyl)-5,6,7-trimethoxy)inden-1-
two recent potent clinically used inhibitors [7], anastro- one) on both human and equine aromatases, in placen-
zole (Arimidex) [8] and letrozole (Femara) [9]. The ®rst tal and testicular microsomes, respectively. The com-
generation inhibitor aminoglutethimide and second parison of these enzymes and their di€erential
and third generation inhibitors, like formestane and interactions with the indane derivative MR 20814 [30],
fadrozole, were found to inhibit in vivo aromatization shown by the kinetic results and molecular modeling,
by 85±93%. The novel aromatase inhibitors letrozole suggested that the amino group of this molecule could
and anastrozole inhibit in vivo aromatization by 97± occupy the entrance of an extrahydrophobic pocket
99%. However, the response rate or the duration of located in the active site according to actual models
remission with these drugs are far from satisfactory [34,45,46]. Moreover, Laughton et al. [45] postulated
[10]. Indeed, in phase III clinical trials, fadrozole, vor- that the alkylation of the glutarimide nitrogen of 3-
ozole, anastrozole, 4-OHA and letrozole caused overall alkyl-3(4-pyridyl)piperidine-2,6-dione led to a better in-
response rates of only 14±16% [11,12], 17% [13], 24± hibition of the aromatase activity by increasing the in-
27% [8], 33% [12] and 36% [14], respectively. Many teraction with the extrahydrophobic pocket. These
studies have currently been undertaken to evaluate sev- previous results allowed us to orientate the synthesis
eral new families of in vitro aromatase inhibitors [15± of a new family of indane derivatives among which
28] illustrating the considerable resources invested in MR 20496 (3-phenyl-2-(pyridin-4-ylmethylen)indan-1-
the development of drugs that may provide a better re- one) was the most potent with an IC50 value of
sponse rate from patients. The need for clinical drugs 0.8 20.1 mM [47]. To check the existence of the extra-
with increased speci®city, clinical eciency and toler- hydrophobic pocket, we postulated that the phenyl
ability remains a challenge in the development of new group of MR 20496 could occupy it within the aroma-
compounds [12,29]. tase active site, and this could reinforce the interaction
Recently, we used a new approach [30] based on the between a nitrogen atom of the inhibitors and the
molecular modeling of the enzyme active site to design heme iron atom of the enzyme. This hypothesis and all
new inhibitors. This was helped, for the ®rst time, by our previous results led us to develop a new family of
the comparison of two mammalian enzymes very well molecules belonging to the indolizinone series, that we
characterized at the biochemical level and cloned: the consider to be a structural compromise between the
human [31±35] and equine [36±44] aromatases. As a benzocycloalkenes-type [48] and the `azole'-type (like
matter of fact, we compared the inhibition e€ects of fadrozole and vorozole) aromatase inhibitors. Some of
 P. Auvray et al. / Journal of Steroid Biochemistry and Molecular Biology 70 (1999) 59±71 61

the compounds tested herein appeared to be more amount; 0.135 g, 0.4 mmol) in water (3 ml) was added
potent than 4-OHA and comparable in vitro to fadro- to a solution of 6-(4-chlorophenyl)-5,6,7,8-tetrahy-
zole. This new family could thus provide information droindolizin-8-one 3 (1 g, 4 mmol) and pyridine-4-car-
concerning the mammalian aromatase active site, and boxaldehyde (0.47 g, 4.4 mmol) in methylene chloride
consequently be of interest for the design of new in- (40 ml), was added. The reaction mixture was stirred
hibitors in estrogen-dependent cancer treatment at room temperature for 2 h. After addition of methyl-
(Scheme 1). ene chloride (80 ml), the organic layer was separated,
washed with water, dried over calcium chloride and
evaporated to dryness. A silica-gel column was used to
2. Materials and methods purify the residue with cyclohexane/ethyl acetate (70/
30) as eluent.
2.1. Materials
2.2.2. (Z ) 6-(4-chlorophenyl)-7-(pyridin-4-
All chemical products were obtained from Sigma (St ylmethylene)-5,6,7,8-tetrahydroindolizin-8-one 4 (MR
Quentin Fallavier, France) or GibcoBRL (Cergy 20492)
Pontoise, France). Polyethylenimine was prepared in Yellow crystals (65%), mp 2388C, IR (KBr) 1665
ddH2O at 10 mM, pH 7.0. [1b,2b-3H]-androstenedione cmÿ1 (CO), 1H NMR (400 MHz, CDCl3) 8.50 (d,
was from Dupont NEN (Les Ulis, France), Coomassie J = 5.6 Hz, 2H, H2 ' and H6 '), 7.94 (s, 1H, Ha), 7.26
brilliant blue G-250 dye from BioRad (Ivry/Seine, (d, J = 8.0 Hz, 2H, H30 and H50), 7.17 (m, 1H, H3),
France), solvents from Carlo Erba (Val de Reuil, 7.10 (m, 4H, H3 ', H5 ', H20 and H60), 6.72 (m, 1H,
France) and from sds (Peypin, France), human embry- H1), 6.25 (m, 1H, H2), 4.59 (m, 1H, H6), 4.45 (dd,
onal kidney 293 cells (ECACC number: 85120602) and J = 12 and 3.5 Hz, 1H, H5a), 4.31 (dd, J = 12 and
human cervix epithelial carcinoma Hela cells (ECACC 2.5 Hz, H5b), 13C NMR (CDCl3) 176.2 (C-8), 150.1
number: 93021013) from CERDIC (Sophia-Antipolis, (C-2 ' and C-6'), 142.6 (C-4 '), 138.9 (C-7), 137.1 (C-10),
France), JM109 bacterial strain from Promega 133.4 (C-8a), 130.6 (C-a), 129.1 (C-20 and C-60), 128.5
(CharbonnieÁres, France), culture media from (C-30 and C-50), 128.2 (C-40), 126.8 (C-3), 123.2 (C-3 '
BioWhittaker (Gagny, France), Thermo Sequenase Kit and C-5 '), 116.5 (C-1), 111.8 (C-2), 50.8 (C-5), 42.1
from Amersham (Les Ulis, France), pCMV plasmid (C-6), analysis calculated for C20H15N2OCl: C, 71.74,
from Invitrogen (NV Leek, The Netherlands) and H, 4.51, N, 8.36, found: C, 71.59, H, 4.52, N, 8.19.
Qiagen Plasmid Maxi Kit from Qiagen (Courtaboeuf,
France). Human aromatase cDNA was kindly pro- 2.2.3. (Z ) 6-(4-chlorophenyl)-7-(pyridin-3-
vided by E.R. Simpson (Dallas, USA). The oligo- ylmethylene)-5,6,7,8-tetrahydroindolizin-8-one 5 (MR
nucleotide primers sense A (5 '-555CCATTGACG 20494)
TCAATGGGAGTT575-3 ') and antisense B (5 '- Yellow oil (45%), IR (KBr) 1670 cmÿ1 (CO), 1H
1169
TAAGGCTTTGCGCATGACCAAG1148-3 ') were NMR (400 MHz, CDCl3) 8.48 (s, 1H, H2 '), 8.44 (m,
from EUROBIO (Les Ulis, France). 1H, H6 '), 7.93 (s, 1H, Ha), 7.45 (d, J = 4.0 Hz, 1H,
H4 '), 7.20 (m, 3H, H5 ', H30 and H50), 7.11 (m, 1H,
2.2. Chemistry H3), 7.05 (d, J = 7.5 Hz, 2H, H20 and H60), 6.63 (m,
1H, H1), 6.18 (m, 1H, H2), 4.53 (m, 1H, H6), 4.39
Melting points were determined on a Ko¯er block (dd, J = 12 and 3.6 Hz, 1H, H5a), 4.24 (dd, J = 12
and are uncorrected. IR spectra were recorded on a and 2.4 Hz, H5b), 13C NMR (CDCl3) 176.5 (C-8),
Unicam Mattson 1000 spectrometer. NMR spectra 150.1 (C-6 '), 149.7 (C-2 '), 139.0 (C-7 and C-4 '), 136.3
(1H, 13C, 1H-COSY, NOE) were recorded at 400 or (C-10), 135.7 (C-3 '), 133.4 (C-8a), 130.7 (C-a), 129.3
100 MHz with tetramethylsilane as an internal stan- (C-20 and C-60), 128.6 (C-30, C-40 and C-50), 126.6 (C-
dard using a JEOL JNM-LA 400 spectrometer. Mass 3), 123.4 (C-5 '), 116.3 (C-1), 111.6 (C-2), 50.9 (C-5),
spectra were recorded on a JEOL D300 instrument 42.2 (C-6), analysis calculated for C20H15N2OCl: C,
using direct inlet system and electron impact ioniz- 71.74, H, 4.51, N, 8.36, found: C, 71.67, H, 4.39, N,
ation. Silica gel 60 (70±230 mesh) was used for column 8.25.
chromatography. Elemental analyses (C, H, N) were The synthesis of other compounds used in this study
performed by INSA, Rouen-France, and agreed with have previously been described [30,47].
the proposed structures within 20.3% of the theoreti-
cal values. 2.3. Preparation of microsomes

2.2.1. Preparation of 4 and 5 (general method) Human placental and equine testicular microsomes
A solution of sodium hydroxide (0.48 g, 12 mmol) were prepared as previously described [30,37]. Brie¯y,
and tetrabutylammonium hydrogen sulfate (catalytic in each instance the available tissue presenting the
62 P. Auvray et al. / Journal of Steroid Biochemistry and Molecular Biology 70 (1999) 59±71

highest speci®c aromatase activity was utilized. Fresh was added to the incubation and kept at 48C for 5
tissue was washed with 0.50 M KCl, was ®rst hom- min. The solution was then centrifuged for 10 min at
ogenized in 50 mM phosphate bu€er pH 7.5 contain- 350g. Aromatase activity was assayed with 300 ml of
ing 0.25 M sucrose, 1 mM DTT, and 4 mM the aqueous phase and labeled androstenedione as
androstenedione in order to preserve the enzyme active described above.
site, and then centrifuged at 20,000g. The supernatant
was further ultracentrifuged at 100,000g and the ®nal 2.7. Spectral studies
pellet was dissolved in the same bu€er containing 20%
glycerol, 1 mM DTT, 0.2 mM EDTA-4 Na, 4 mM Absorbance of microsomal proteins (1.5 mg) in 1.5
androstenedione, and stored at ÿ808C until use. ml of 50 mM Tris±Maleate, pH 7.4 was measured with
Protein concentration was evaluated according to a Kontron±Uvikon 860 spectrophotometer. Di€erence
Bradford [49] using bovine serum albumin as standard, absorption spectra were recorded at 378C over 350±
and Coomassie brilliant blue as dye-reagent. 500 nm after addition of 100 mM of each inhibitor
diluted in ethanol (16 ml). The spectrum of ethanol
2.4. Inhibition studies with microsomes alone was comparable to the baseline (350±500 nm).
The spectra of the microsomes and of the molecule
Aromatase activity was evaluated by measuring alone were subtracted from the spectrum of micro-
3
H2O released from 200 nM [1b,2b-3H]-androstene- somes plus molecule.
dione (speci®c activity: 1554 GBq/mmol) at 378C for
15 min [30,32], in the presence of various inhibitors 2.8. pCMV-human aromatase cDNA construction
from 0 to 15 mM. Reactions with microsomes (195 mg
and 20 mg of human and equine microsomal proteins, Human aromatase cDNA (2920 bp) [33] was pre-
respectively for determination of IC50 values) were in- viously cloned into pUC18 (2.7 kb) with two frag-
itiated by adding 60 mM NADPH,H+ to a ®nal ments, lgt10 HindIII±EcoRI in 5 ' end (240 bp) and
volume of 0.5 ml and stopped by adding 1 ml of EcoRI±BglII in 3 ' end (900 bp). This construction was
chloroform. Steroids were then extracted by incubation partially digested with EcoRI and the 2920 bp frag-
with a charcoal/dextran solution (7%/1.5%) and the ment EcoRI±EcoRI subcloned into the pCMV EcoRI
radioactivity of the aqueous phase was measured as site. Orientation was then checked by PCR ampli®ca-
previously described [37]. Control incubations were tion with the primers sense A and antisense B which
realized by incubating microsomes, substrate and in- are speci®c for pCMV and the cDNA, respectively (the
hibitors without NADPH,H+ in the same conditions. amplicon length was 1359 bp) and by sequencing. The
Results were the mean of triplicate experiments 2 S.D. pCMV±cDNA was puri®ed from transformed JM109
and were expressed as pmol estrogen formed/min. mg bacterial strain by using the Qiagen Plasmid Maxi Kit.
microsomal proteins. The length, the concentration and the purity of the
plasmid-cDNA construction were veri®ed by 1% agar-
2.5. Kinetic studies ose electrophoresis and ethidium bromide staining.

Concentration range was 0±600 nM for inhibitors 2.9. 293 cells culture and transfection
and 6±100 nM for substrate. Aromatase activity was
evaluated at the linear portion of the Michaelis± Human embryonal kidney 293 cells were grown at
Menten plot by incubating 5 mg (human) or 4 mg 378C in red phenol free EMEM medium supplemented
(equine) microsomal proteins with substrate and var- with 2 mM glutamine, 10% new born calf serum
ious inhibitors, at 378C for 12 min with human aroma- (supreme serum), 1% nonessential amino acid under
tase and 8 min with the equine enzyme. Km and Ki an atmosphere of 5% CO2 and 95% air. Fifty thou-
values were determined graphically by using, respect- sand cells were grown to 50% con¯uence on 24-wells
ively, Lineweaver±Burk and Dixon representations. cell culture plates 18 h before transfection, washed
with serum-free cell culture medium, supplemented
2.6. Inactivation studies with 500 ml serum-free medium and transiently trans-
fected with 2 mg pCMV±human aromatase cDNA
Inactivation studies were carried out with 26 mg using a modi®cation of the method of Boussif et al.
(human) or 20 mg (equine) microsomal proteins by [50]. Brie¯y, 2 mg pCMV±cDNA (corresponding to 6
testing di€erent incubation times (0±30 min) at 258C nmol of phosphate) and 54 nmol of polyethylenimine
with 10 mM inhibitors and 60mM NADPH,H+ in 0.1 were separately diluted with 50 ml 150 mM NaCl, incu-
M Na-phosphate bu€er, pH 7.5, containing 0.5 M bated 10 min at room temperature under laminar fume
sucrose according to Moslemi and SeÂralini [42]. An hood, mixed together, then further incubated 10 min
activated charcoal-dextran mixture (2%/1%, 100 ml) at room temperature and added to each well. Cells
 P. Auvray et al. / Journal of Steroid Biochemistry and Molecular Biology 70 (1999) 59±71 63

were incubated 3±4 h at 378C and then supplemented

with 500 ml medium containing 10% supreme serum.
After a further 18 h incubation, cells were washed with
serum-free medium and the aromatase activity
measured `in cell'.

2.10. Aromatase activity and inhibition ``in cell''

In this study, we evaluated aromatase activity `in

cell' according to Zhou et al. [51] by measuring 3H2O
released from 200 nM [1b,2b-3H]-androstenedione.
Brie¯y, cells were washed with serum-free culture med-
ium. The dessicated radioactive substrate supplemented
with 1 mM progesterone (used to inhibit the possible
endogenous 5a-reductase activity) and 0±10 mM inhibi-
tor, were mixed with serum-free culture medium and
added to each well. Cells were incubated at 378C
under 5% CO2 atmosphere for 45 min. After incubat-
ing culture plates 5 min on ice, 1 ml culture medium
was sampled and mixed with 1 ml chloroform.
Steroids were extracted by incubation with a 1 ml
charcoal dextran solution (7%/1.5%) and the radioac-
tivity of the aqueous phase was measured as previously
described [37]. Control incubation was realized by
transfecting in the same conditions the pCMV plasmid
alone instead of the pCMV±cDNA plasmid. The
results were the mean of triplicate experiments 2 S.D.
and were expressed as the percentage of control.

2.11. Hela cells culture and cytotoxicity study

Fig. 1. Inhibition of human (A) and equine (B) aromatases by MR
Hela cells were grown at 378C in red phenol free 20492 and MR 20494 in comparison with 4-OHA and fadrozole.
EMEM medium in the same conditions previously Aromatase activity was evaluated by measuring 3H2O released from
200 nM [1b,2b-3H]-androstenedione at 378C for 15 min, in the pre-
described. In the trypan blue exclusion test, 100,000 sence of inhibitors MR 20492 and MR 20494 and fadrozole or 4-
cells were plated on day 0 and grown to 50% con¯u- OHA used as controls. The inhibition of aromatase in human pla-
ence on six-wells cell culture plates, treated (day 1) cental (195 mg) and equine testicular (20 mg) microsomes was per-
with 10 mM inhibitor during 24, 48 and 72 h (fresh cul- formed in the presence of 60 mM NADPH,H+. The blank was
ture medium containing 10 mM inhibitor was added realized without adding NADPH,H+. The results are expressed as
percentage to a standard control which was incubated with
daily). Cells were harvested by 0.25% trypsin-EDTA NADPH,H+ and without inhibitor, and are the mean of triplicate
digestion, treated with trypan blue and counted using experiments2 S.D.
a hematocytometer. The results were the mean of tri-
plicate experiments 2 S.D. and were expressed as cells/
ml culture medium. In the MTT (3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, the substrate MTT by the mitochondrial succinate-de-
50,000 cells were plated on 24-wells cell culture plates hydrogenase. This solution was then removed and
and treated in the same conditions as previously added to 1 ml PBS before being evaluated by spectro-
described for the trypan blue exclusion test. The photometer with MTT as reference (this reference was
method of Denizot and Lang [52] was adapted to our incubated in the same conditions of the assay but with-
model. Brie¯y, MTT (5 mg/ml in PBS) was diluted to out cells). The wavelengths were determined for our
1 mg/ml in culture medium. Culture supernatant was model (see Results and the legend of Fig. 6).
then removed by inverting, ¯icking and blotting the
plate. MTT (250 ml) solution was added to the cells, 2.12. Statistical study
gently shaken and incubated 3 h at 378C. The plates
were ®nally inverted, ¯icked and blotted to remove the A t-test with unpaired values was performed with
untransformed MTT. DMSO (250 ml) was added to the Statview demo program version 4.5.3 (Abacus
dissolve the formazan formed by the transformation of Concepts).
64 P. Auvray et al. / Journal of Steroid Biochemistry and Molecular Biology 70 (1999) 59±71

Table 1
Human (H) and equine (E) aromatase inhibition with the compounds used in this study. 4-OHA, fadrozole and MR 20814 are indicated for com-
parison. Km values with androstenedione were 10.7 and 6.2 nM for human and equine enzymes, respectively. See legends of Figs. 1 and 2 for
details

Compounds IC50 (mM) Ki (nM) Ki/Km

H E H E H E

Fadrozole 0.05920.029 0.28320.161 0.620.4 2.520.0 0.120.0 0.4 20.0

4-OHA 0.45020.057 0.58020.243 20.228.8 6.220.0 1.920.8 1.0 20.0
MR 20814 3.49321.167 >10 86.227.8 577.7236.9 8.120.7 93.9 26.0
MR 20492 0.14720.013a,c 1.28 20.002a,b,c 10.323.3a,c 58.3 24.2a,b,c 1.020.3a,c 9.0 20.6a,b,c
MR 20494 0.11120.063c 1.34120a,b,c 5.522.6a,c 39.4 210.6a,b,c 0.520.3c 6.1 21.6a,b,c

a
p < 0.05 compared to fadrozole.
b
p < 0.05 compared to 4-OHA.
c
p < 0.05 compared to MR 20814.

3. Results

3.1. Chemistry

The synthesis of the aromatase inhibitors was

achieved starting either from indanones or from indoli-
zinones. Involvement of baclofen 1 in a Clauson±Kaas
reaction led to pyrrolylbaclofen 2 which was sub-
sequently cyclized under Vilsmeier conditions into the
phenyltetrahydro indolizinone 3 [53]. The ®nal tested
compounds 2±6 were some pyridinyl derivatives result-
ing from an aldolization reaction run from the indoli-
zinone 3. On the other hand, reaction of 3 with
pyridinecarboxaldehyde, in a heterogenous system
(dichloromethane/aqueous sodium hydroxide solution)
and in the presence of tetrabutyl-ammonium sulfate as
catalyst, yielded the pyridinylmethylene-indolizinones
4, 5 (MR 20492 and MR 20494, respectively). The
assignment of the Z con®guration of 4 was made on
the basis of NOE experiments. Speci®cally, irradiation
of the methylene proton in 4 led to a NOE with both
H-2 ' and H-6 '. Furthermore 4, in d6-DMSO solution
su€ered, after a week at room temperature, a partial
isomerization into its E form whose 1H NMR spec-
trum exhibited a shielded methylene signal at 6.88 ppm
(7.80 ppm for the Z form) con®rming the initial con-
®guration. Con®gurations of 5 were determined by
analogy between their 1H NMR spectra and those of
4.

3.2. Biological results on human aromatase in placental

microsomes Fig. 2. Kinetic inhibition studies with MR 20492 (A) and MR 20494
(B) on human aromatase. Aromatase activity was evaluated by incu-
In this study, we tested the inhibitory e€ect of newly bating 5 mg of placental microsomal proteins with [1b,2b-3H]-andros-
synthesized indolizinone derivatives on the human tenedione (6±100 nM) and with 0 to 60 nM of each compound at
378C during 12 min. Ki were determined graphically by Dixon-plot.
aromatase activity. We obtained two potent molecules
Results are expressed as pmol estrogen formed/min.mg microsomal
4 (MR 20492) and 5 (MR 20494) that inhibited aroma- proteins and are the mean of triplicate values2 S.D. Results are
tase (Fig. 1A) with an IC50 of 0.147 20.013 mM and representative of one from three experiments showing similar pro-
0.111 2 0.063 mM, respectively (Table 1). The IC50 ®les.
 P. Auvray et al. / Journal of Steroid Biochemistry and Molecular Biology 70 (1999) 59±71 65

Fig. 3. Preincubation tests of human (A) and equine (B) aromatases

with MR 20492, MR 20494 or 4-OHA. Microsomal proteins from
human placenta (26 mg, (A)) or equine testes (20 mg, (B)) were incu-
bated with 10 mM of MR 20492, MR 20494 or 4-OHA and 60 mM Fig. 4. Kinetic inhibition studies with MR 20492 (A) and MR 20494
NADPH,H+ at 258C during 0±30 min. 4-OHA was used as control. (B) on equine aromatase. Testicular microsomal proteins (4 mg) were
Incubations were stopped by adding a charcoal/dextran mixture incubated with 6±100 nM [1b,2b-3H]-androstenedione and with 0±
(2%/1%) and after centrifugation aromatase activity was assayed 600 nM MR 20492 (A) or MR 20494 (B) at 378C during 8 min.
with 300 ml of the aqueous phase. Results are expressed as % to a Results are expressed as pmol estrogen formed/min mg microsomal
standard control which was incubated with NADPH,H+ and with- proteins and are the mean of triplicate values2 S.D. Results are
out inhibitor and are the mean of triplicate experiments2 S.D. representative of one from three experiments showing similar pro-
®les.
value for MR 20494 was not signi®cantly di€erent
from that obtained for fadrozole (0.059 20.029 mM), for fadrozole (Ki/Km=0.1 2 0.0). Furthermore, Ki/Km
which is currently in phase III clinical trials, and both values for both indolizinone derivatives were 2 to 4-
indolizinone derivatives have an inhibitory potency 3 fold lower than for 4-OHA (1.9 20.8). However, these
to 4-fold greater than 4-OHA (0.4502 0.057 mM), compounds presented an inhibitory potency greater
which is clinically available. Moreover, MR 20492 and than MR 20814.
MR 20494 were 24 to 31 times more potent than the
indane derivative MR 20814 (3.4932 1.167 mM). A 3.3. Biological results on equine aromatase in testicular
kinetic study was performed and is summarized in microsomes
Table 1. These new indolizinone derivatives presented
a competitive inhibition (Fig. 2) but they are not inac- In order to understand better the active site of
tivators in contrast to 4-OHA (Fig. 3A). Furthermore, human aromatase by comparative study and to design
we showed that compounds MR 20492 and MR 20494 new more ecient inhibitors, we tested the indolizi-
were very potent inhibitors since their Ki/Km ratio none derivatives on the equine enzyme (Fig. 1B). The
values, which represented their relative inhibitory IC50 values obtained were 1.28 2 0 mM and 1.341 2 0
potency, were 1.0 20.3 and 0.5 2 0.3, respectively. mM with MR 20492 and MR 20494, respectively,
However, these results were slightly higher than those which were lower than the values evidenced for MR
66 P. Auvray et al. / Journal of Steroid Biochemistry and Molecular Biology 70 (1999) 59±71

Fig. 6. Conformations of the Z (left) and E (right) forms for the

compound 15 (MR 20492, R con®guration). The conformations were
obtained with the Discover software (MSI). Optimization of the geo-
metry was carried out with the steepest descents and conjugate gradi-
ent methods (CVFF force ®eld) with a gradient of 0.01 kcal/(mol/AÊ)
for the convergence criteria.

[30], we did not evidence a type I or type II spectrum

for MR 20492 and MR 20494 but another spectrum
characterized by a single absorbance maximum at
400±410 nm which characterizes, in the same manner,
both human (Fig. 5) and equine (data not shown)
enzymes.

3.5. Aromatase inhibition in cell culture

In this experiment, we tested inhibitory potencies of

indolizinone in cell cultures, in comparison with MR
20814 [30], fadrozole and 4-OHA used as known
potent inhibitors of aromatase. MR 20492 and MR
20494 were tested in human embryo kidney 293 cells
Fig. 5. Spectral studies of interactions between MR 20492 (A) or transfected with human aromatase cDNA. As shown
MR 20494 (B) and the human aromatase active site. Absorbance
from 350 to 500 nm of microsomal proteins (1.5 mg) with 100 mM of
in Table 2, MR 20494, like fadrozole and 4-OHA,
inhibitor was measured at 378C. The spectra of the microsomes or inhibited aromatase activity in cells in the same order
inhibitor alone were subtracted from the spectrum measured during of magnitude as was observed for microsomes (for
the incubation of microsomes with the chosen molecule. Results are microsomes, IC50=3.5 21.2 mM for MR 20814; Table
representative of one from three experiments showing similar pro- 1). However, MR 20492 and MR 20814 were slightly
®les. The interactions between MR 20492 or MR 20494 and the
active site of equine aromatase gave essentially similar spectra (data
not shown).
Table 2
Inhibition of human aromatase activity in 293 cells transfected with
20814, but higher than the values obtained for 4-OHA human aromatase cDNA. 50,000 cells in 24-wells plates were trans-
and fadrozole (0.580 20.243 mM and 0.283 2 0.161 fected with 2 mg pCMV-human aromatase cDNA with polyethyleni-
mine as transfecting agent [55]. Aromatase activity was evaluated by
mM, respectively), and much higher than values
measuring 3H2O released from 200 nM [1b,2b-3H]-androstenedione
obtained with the human enzyme. The di€erential in- incubated in culture medium at 378C±5% CO2 atmosphere for 45
hibitory potencies were reinforced by Ki/Km ratio min in the presence of di€erent inhibitors. Results are expressed as
values which are 9 and 12 times greater (for MR % 2S.D. to a standard control which was incubated without inhibi-
20492 and MR 20494, respectively) than those tor in the same conditions and are the mean of four experiments
obtained with human aromatase (Table 1). As
Compounds IC50 (mM)
observed for the human enzyme, these two inhibitors
were not inactivators of the equine enzyme (Fig. 3B) Fadrozole 0.20 20.02
and led to a competitive inhibition (Fig. 4). 4-OHA 0.50 20.40
MR 20814 104520.60a
MR 20494 0.20 20.03
3.4. Spectral study on human and equine aromatases
MR 20492 3.90 20.93a

In this context, in contrast with our previous results a

p < 0.05 compared to IC50 obtained with microsomes.
 P. Auvray et al. / Journal of Steroid Biochemistry and Molecular Biology 70 (1999) 59±71 67

Fig. 7. Cytotoxicity study of new human aromatase inhibitors on Hela cells. 50,000 cells in 24-wells plates were treated with 10 mM inhibitors
during 24, 48 or 72 h. (A) The MTT assay was adapted to Hela cells and the formazan production was performed at 24, 48 and 72 h between
405 and 690 nm (the MTT was used as reference). (B) The formazan spectrum was evidenced at 72 h of cell culture and two wavelengths were
evaluated: 565 and 750 nm for the test and reference, respectively. (C) Cytotoxicity was evaluated by measuring the transformation of the mito-
chondrial succinate-dehydrogenase substrate (MTT) in blue formazan product. The absorbance at reference wavelength was subtracted from the
test absorbance and results were the mean of triplicate values2S.D. Fadrozole, 4-OHA, MR 20814 and MR 20818 [35] were also used as con-
trols. T corresponded to cells without inhibitor. No absorbance was signi®cantly di€erent compared to the control T.

less potent (3.9 2 0.9 mM and 10.4 20.6 mM, respect- not lead to cellular death with this method and even at
ively). a concentration of 10 mM no cytotoxicity was evi-
denced, either with indane derivatives or with indolizi-
3.6. Cytotoxicity in cell culture none derivatives on Hela cells during culture times of
24, 48 or 72 h. In fact, cells were fast growing up to 48
The results obtained with the 293 cells revealed that h and more moderately until 72 h. This method was
the molecules MR 20492 and MR 20494 were not modi®ed by using a spectrophotometer and optimized
cytotoxic since no dead cell was observed in this experi- to our Hela cells by testing di€erent wavelengths
ment. Moreover, we tested the cytotoxicity of these during 24, 48 and 72 h of culture. As shown in Fig.
compounds, as well as the potent indane derivative in- 7A, the absorbance increased proportionally to the
hibitor MR 20814, on the human cervix epitheloid car- time of cell culture. Moreover, Fig. 7B allowed deter-
cinoma Hela cells by using the trypan blue exclusion mination of the test wavelength (565 nm) and reference
test (data not shown). In this type of study, the usual wavelength (750 nm). As the maximal absorbance of
concentration of inhibitor which is supposed to not DMSO was at 503 nm [55], it did not interfere with
a€ect cell survival is 1 mM [54]. This concentration did the chosen test wavelength. The results obtained with
68 P. Auvray et al. / Journal of Steroid Biochemistry and Molecular Biology 70 (1999) 59±71

the MTT assay (Fig. 7C) con®rmed our previous benzoylpyridine showed exclusively a reverse type I
results since none of the tested molecules showed a spectrum and pyridine and its derivatives substituted
cytotoxic e€ect. at the 3- or 4-positions with phenyl or benzoyl groups
showed exclusively a type II spectrum. By considering
the Z form for the compounds MR 20492 and MR
4. Discussion 20494, the type II spectrum should be obtained.
However, if the E form and the R con®guration are
In this study, we screened the e€ect of indolizinone considered, the pyridinic nitrogen is sterically hindered,
derivatives on human and equine aromatases in pla- by the chlorophenyl group, from access to the heme
cental and testicular microsomes, respectively. We iron. This situation could lead to a reverse type I spec-
obtained two molecules, MR 20492 and MR 20494, trum (see Fig. 6). The isomerization of the compound
which are very potent inhibitors. Indeed, IC50 and Ki/ MR 20492 (or MR 20494) was checked and indeed, in
Km values obtained for MR 20494 evidenced that this polar solvent, this fact was observed leading to a mix-
molecule was 694 times more potent than aminoglu- ture of E and Z forms explaining the pro®le for the
tethimide and slightly less potent than fadrozole which UV spectra obtained during the conditions of the bio-
is in phase III clinical trials. However, MR 20494 and logical evaluations. Thus, we suggest that the inhi-
MR 20492 were not inactivators. Moreover, our com- bition results are due to a combination of both
parative results between models also con®rm confor- geometric isomer activities. However, we previously
mational di€erences between the active sites of human showed with indane derivatives [30] that an inhibitor
and equine aromatases. Indeed, as described in our characterized by an interaction with the heme iron
previous work [30], we suggested that the chlorophenyl (type II spectrum) was more potent. We thus suggest
group could interact with the extrahydrophobic pocket that MR 20492 and MR 20494 could be more ecient
within the active site and that the N atom of the pyri- if the isomer proportion tended to the `type II spec-
dyl group could be close to the heme iron. The inter- trum' one.
action of these new inhibitors with this pocket could In the present study, we evidenced in vitro two
be stronger with the human aromatase than the inter- potent inhibitors of human aromatase in placental
action previously described [30] with the amino group microsomes, MR 20492 and MR 20494. As our mol-
of MR 20814, and could then explain the higher inhi- ecules could also be developed as drugs, it would be
bition observed. Moreover, with the equine aromatase, useful to evaluate their ability to penetrate cells. As
the chlorophenyl group of MR 20492 and MR 20494 shown in Table 2, MR 20494 seemed to be an interest-
could strongly interact with the `acidic pocket' pre- ing candidate since it remained highly potent in cell
viously described [30] and formed by D309, D476 and cultures like fadrozole. These results suggest that MR
N475. This interaction could remove the pyridyl group 20494, more easily than MR 20492, was able to pass
from the heme iron and then render the inhibitor less through the cellular membrane, suggesting that it was
potent in comparison to the human enzyme. not or little metabolized before reaching its speci®c
The study of the interaction between the heme iron target.
and a substrate or an inhibitor has currently been con- Moreover, in order to evaluate the cytotoxicity of
ducted with cytochromes P450 [56], as for example these compounds, we adapted the MTT assay to our
with aromatase [30,31,57,58]. In our previous study, a model. Although the trypan blue exclusion test is cur-
type II spectrum, considered as speci®c for an inter- rently used in cytotoxicity studies, this method only
action between nitrogen atom of the molecule and the revealed membrane permeability and not the intra-
heme iron of the cytochrome, with minimal absor- cellular damages [60]. Mosmann [61] then developed a
bance at 390 nm and maximal absorbance at 420 nm, quantitative colorimetric assay to detect mammalian
was observed between the indane derivative 3-amino- cell survival and proliferation based on the tetrazolium
5-methoxy-2-(pyridin-4-ylmethyl)inden-1-one (MR salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tet-
20814) and human aromatase, whereas a type I spec- razolium bromide). This assay detects living, but not
trum (inverted absorbance) was observed for equine dead cells and the signal generated is dependent on the
aromatase. In this study, the spectra produced by the degree of activation of the cells since the tetrazolium
interaction between MR 20492 or MR 20494 and the ring is cleaved by active mitochondria. This method
active site of both enzymes showed an unexpected and was validated by comparison with the [3H]-thymidine
unique type spectrum since it was characterized by an uptake method [52], and Carmichael et al. [55] demon-
absorbance peak at 400±410 nm. One hypothesis to strated that it was a fast and ecient method for
explain this pro®le is that a combination of type I and studying the cytotoxicity of compounds in cell culture.
type II spectra was obtained. A previous study showed Mosmann [61] previously determined a test wavelength
the possibility of getting two di€erent types of spectra of 570 nm and a reference wavelength of 630 nm (this
in relation to the substituents on pyridine [59]. The 2- value correlates with the baseline) whereas Denizot
 P. Auvray et al. / Journal of Steroid Biochemistry and Molecular Biology 70 (1999) 59±71 69

and Lang [52] used 560 nm test and 690 nm reference [7] New aromatase inhibitors for breast cancer. Drug Ther. Bull.,
wavelengths, and Carmichael et al. [55] a 540 nm test 35 (1997) 55±56.
[8] A.U. Buzdar, S.E. Jones, C.L. Vogel, J. Wolter, P. Plourde, A.
wavelength when using DMSO as solvent. All these Webster, The Arimidex Study Group, A phase III trial com-
authors used this MTT assay by reading on an paring anastrozole (1 and 10 mg), a potent and selective
enzyme-linked immunosorbent assay reader and with aromatase inhibitor, with megestrol acetate in postmenopausal
di€erent cells. Our results, using a test wavelength at women with advanced breast carcinoma, Cancer 79 (1997)
565 nm and a reference wavelength at 750 nm, allowed 730±739.
[9] J.N. Ingle, P.A. Johnson, V.J. Suman, J.B. Gerstner, J.A.
us to show that neither MR 20492 nor MR 20494 Mailliard, J.K. Camoriano, H. GesmeD, C.L. Loprinzi, A.K.
were cytotoxic after 72 h incubation with Hela cells. Hat®eld, L.C. Hartmann, A randomized phase II trial of two
In conclusion, these results are promising with dosage levels of letrozole as third-line hormonal therapy for
regard to the development of new drugs as we evi- women with metastatic breast carcinoma, Cancer 80 (1997)
denced two new original inhibitors which are able to 218±224.
[10] P.E. Lonning, Aromatase inhibition for breast cancer treat-
strongly inhibit human aromatase in placental micro- ment, Acta Oncol. 35 (1996) 38±43.
somes and in cell culture. They allow a better modeling [11] R.C. Stein, M. Dowsett, J. Davenport, A. Hedley, H.T. Ford,
and understanding of the aromatase active site by J.C. Gazet, R.C. Coombes, Preliminary study of the treatment
comparison with the equine enzyme, and this new de- of advanced breast cancer in postmenopausal women with the
sign will be tested by site-directed mutagenesis studies. aromatase inhibitor CGS 16949A, Cancer Res. 50 (1990) 1381±
1384.
It would be interesting to examine the e€ects of MR [12] W.R. Miller, Aromatase inhibitors: where are we now?, Br. J.
20492 and MR 20494 on other steroidogenic enzymes, Cancer 73 (1996) 415±417.
and in vivo. [13] S.R. Johnston, I.E. Smith, D. Doody, S. Jacobs, H.
Robertshaw, M. Dowsett, Clinical and endocrine e€ects of the
oral aromatase inhibitor Vorozole in postmenopausal patients
with advanced breast cancer, Cancer Res. 54 (1994) 5875±5881.
Acknowledgements [14] G. Bisagni, G. Cocconi, F. Scaglione, F. Fraschini, C. P®ster,
P.F. Trunet, Letrozole, a new oral non-steroidal aromastase in-
hibitor in treating postmenopausal patients with advanced
This work was initiated and developed through a breast cancer. A pilot study, Ann. Oncol. 7 (1996) 99±102.
grant from the Ligue Nationale Contre Le Cancer [15] R.W. Hartmann, M. Frotscher, D. Ledergerber, G.A.
(Comite de la Manche) for the biological studies and a Wachter, G.L. Grun, T.F. Sergejew, Synthesis and evaluation
studentship to P.A., and recently supported by the of azole-substituted tetrahydronaphtalenes as inhibitors of
P450 arom, P450 17, and P450 TxA2, Arch. Pharm. 329 (1996)
Ligue Nationale Contre Le Cancer, Comite du
251±261.
Calvados, for the chemical syntheses. The Conseil [16] M. Kudoh, Y. Susaki, Y. Ideyama, T. Nanya, M. Mori, H.
GeÂneÂral du Calvados and Fonds FEDER supported Shikama, T. Fujikura, Inhibitory e€ect of a novel non-steroidal
the equine studies. We wish to thank Dr. B. Plainfosse aromatase inhibitor, YM511 on the proliferation of MCF-7
for providing horse tissues, M.J. Simon and S.Galopin human breast cancer cell, J. Steroid Biochem. Mol. Biol. 58
(1996) 189±194.
for technical assistance.
[17] D. Lesuisse, J.F. Gourvest, O. Benslimane, F. Canu, C.
Delaisi, B. Doucet, C. Hartmann, J.M. Lefrancois, B. Tric, D.
Mansuy, D. Philibert, G. Teutsch, Structure-activity relation-
ships of a new family of steroidal aromatase inhibitors. 1.
References Synthesis and evaluation of a series of analogs related to 19-
[(methylthio)methyl]androstenedione (RU54115), J. Med.
[1] A.M. Brodie, Overview of recent development of aromatase in- Chem. 39 (1996) 757±772.
hibitors, Cancer Res. 42 (1982) 3312s±3314s. [18] V.C.O. Njar, J. Duerkop, R.W. Hartmann, Novel 19-(cyclo-
[2] M.J. Carella, N.V. Dimitrov, V.V. Gossain, L. Srivastava, propylamino)-androst-4-en-3,17-dione: a mechanism-based in-
D.R. Rovner, Adrenal e€ects of low-dose aminoglutethimide hibitor of aromatase, J. Enzyme Inhib. 10 (1996) 47±56.
when used alone in postmenopausal women with advanced [19] G.A. Wachter, R.W. Hartmann, T. Sergejew, G.L. Grun, D.
breast cancer, Metabolism 43 (1994) 723±727. Ledergerber, Tetrahydronaphthalenes: in¯uence of heterocyclic
[3] L.R. Wiseman, K.L. Goa, Formestane: a review of its pharma- substituents on inhibition of steroid enzymes P450 arom and
cological properties and clinical ecacy in the treatment of P450 17, J. Med. Chem. 39 (1996) 834±841.
postmenopausal breast cancer, Drugs Aging 9 (1996) 292±306. [20] E. Bajetta, N. Zilembo, S. Barni, C. Noberasco, A. Martinetti,
[4] R.J. Santen, P. Langecker, S.J. Santner, S. Sikka, J. Rajfer, R. L. Ferrari, G. Schieppati, R. Buzzoni, A. Jirillo, M.D.
Swerdlo€, Potency and speci®city of CGS-16949A as an Amichetti, M. Aprile, G. Comella, E. Bichisao, G.F. Bolelli, A.
aromatase inhibitor, Endocr. Res. 16 (1990) 77±91. Attili, E. Bombardieri, The Italian Trials in Medical Oncology
[5] P.C. De Jong, J. Van de Ven, H.W. Nortier, I. Maitimu- (I.T.M.O.) group, A multicentre, randomized, pharmacoki-
Smeele, T.H. Donker, J.H. Thijssen, P.H. Slee, R.A. netic, endocrine and clinical study to evaluate formestane in
Blankenstein, Inhibition of breast cancer tissue aromatase ac- breast cancer patients at ®rst relapse: endocrine and clinical
tivity and estrogen concentrations by the third-generation results, Ann. Oncol. 8 (1997) 649±654.
aromatase inhibitor vorozole, Cancer Res. 57 (1997) 2109± [21] J.G. Blanco, R.R. Gil, C.I. Alvarez, L.C. Patrito, S. Genti-
2111. Raimondi, A. Flury, A novel activity for a group of sesquiter-
[6] L.R. Wiseman, C.M. Spencer, Vorozole, Drugs Aging 11 pene lactones: inhibition of aromatase, FEBS Lett. 409 (1997)
(1997) 245±250. 396±400.
70 P. Auvray et al. / Journal of Steroid Biochemistry and Molecular Biology 70 (1999) 59±71

[22] M. Le Borgne, P. Marchand, M. Du¯os, B. Delevoye-Seiller, and kinetic characteristics, Comp. Biochem. Physiol. 100B
S. Piessard-Robert, G. Le Baut, R.W. Hartmann, M. Palzer, (1991) 107±115.
Synthesis and in vitro evaluation of 3-(1-azolylmethyl)-1H- [39] A. Tomilin, E. Komarova, S. Moslemi, P. Auvray, P.
indoles and 3-(1-azolyl-1-phenylmethyl)-1H-indoles as inhibi- Sourdaine, M.A. Drosdowsky, G.E. SeÂralini, Molecular clon-
tors of P450 arom, Arch. Pharm. 330 (1997) 141±145. ing and characterization of equine cytochrome P450 aromatase,
[23] M. Numazawa, M. Oshibe, S. Yamaguchi, 6-Alkylandrosta- in: IV International Aromatase Conference, Tahoe City, CA,
4,6-diene-3,17-diones and their 1,4,6-triene analogs as aroma- 1996 Abst. A22.
tase inhibitors: structure±activity relationships, Steroids 62 [40] J. Almadhidi, G.E. SeÂralini, J. Fresnel, P. Silberzahn, J.L.
(1997) 595±602. Gaillard, Immunohistochemical localization of chromosome
[24] M. Okada, T. Yoden, E. Kawaminami, Y. Shimada, M. P450 aromatase in equine gonads, J. Histochem. Cytochem. 43
Kudoh, Y. Isomura, H. Shikama, T. Fujikura, Studies on (1995) 571±577.
aromatase inhibitors. I. Synthesis and biological evaluation of [41] J. Almadhidi, S. Moslemi, M.A. Drosdowsky, G.E. SeÂralini,
4-amino-4H-1,2,4-triazole derivatives, Chem. Pharm. Bull. 44 Equine cytochrome P450 aromatase exhibits an estrogen 2-hy-
(1996) 1871±1879. droxylase activity in vitro, J. Steroid Biochem. Mol. Biol. 59
[25] M. Okada, T. Yoden, E. Kawaminami, Y. Shimada, M. (1996) 55±61.
Kudoh, Y. Isomura, Studies on aromatase inhibitors. II. [42] S. Moslemi, G.E. SeÂralini, Inhibition and inactivation of equine
Synthesis and biological evaluation of 1-amino-1H-1,2,4-tria- aromatase by steroidal and non-steroidal compounds: a com-
zole derivatives, Chem. Pharm. Bull. 45 (1997) 333±337. parison with human aromatase inhibition, J. Enz. Inhib. 12
[26] M. Okada, T. Yoden, E. Kawaminami, Y. Shimada, M. (1997) 241±254.
Kudoh, Y. Isomura, Studies on aromatase inhibitors. III. [43] S. Moslemi, A. Vibet, V. Papadopoulos, L. Camoin, P.
Synthesis and biological evaluation of [(4-bromobenzyl)(4-cya- Silberzahn, J.L. Gaillard, Puri®cation and characterization of
nophenyl)amino]azoles and their azine analogs, Chem. Pharm. equine testicular cytochrome P-450 aromatase: comparison
Bull. 45 (1997) 482±486. with the human enzyme, Comp. Biochem. Physiol. 118B (1997)
[27] M. Okada, T. Yoden, E. Kawaminami, Y. Shimada, M. 217±227.
Kudoh, Y. Isomura, Studies on aromatase inhibitors IV. [44] S. Moslemi, P. Auvray, P. Sourdaine, M.A. Drosdowsky, G.E.
Synthesis and biological evaluation of N,N-disubstituted-5-ami- SeÂralini, Structure±function relationships for equine and
nopyrimidine derivatives, Chem. Pharm. Bull. 45 (1997) 1293± human aromatases: a comparative study, Ann. NY Acad. Sci.
1299. 839 (1998) 576±577.
[28] M. Takahashi, T. Kayo, T. Karakida, S. Nakagawa, M. Kato, [45] C.A. Laughton, R. Mckenna, S. Neidle, M. Jarman, R.
S. Matsuno, Y. Koide, M. Sakato, S. Kawashima, Potent and McCague, Crystallographic and molecular modeling studies on
selective aromatase inhibitor: in vitro and in vivo studies with 3-ethyl-3-(4-pyridyl)piperidine-2,6-dione and its butyl analogue,
s-triazine derivative SEF19, Endocr. Res. 23 (1997) 1±13. inhibitors of mammalian aromatase. Comparison with natural
[29] A. Howell, S. Downey, E. Anderson, New endocrine therapies substrates: prediction of enantioselectivity for N-alkyl deriva-
for breast cancer, Eur. J. Cancer 32A (1996) 576±588. tives, J. Med. Chem. 33 (1990) 2673±2679.
[30] P. Auvray, S. Moslemi, P. Sourdaine, G.E. SeÂralini, C. [46] D. Zhou, L.L. Cam, C.A. Laughton, K.R. Korzekwa, S. Chen,
Enguehard, P. Dallemagne, P. Sonnet, R. Bureau, S. Rault, Mutagenesis study at a postulated hydrophobic region near the
Evidence for new potent nonsteroidal human aromatase inhibi- active site of aromatase cytochrome P450, J. Biol. Chem. 269
tors and comparison with equine aromatase inhibition for an (1994) 19501±19508.
understanding of the mammalian active site, Eur. J. Med. [47] P. Sonnet, P. Auvray, J. Guillon, P. Dallemagne, R. Bureau, S.
Chem. 33 (1998) 451±462. Rault, S. Moslemi, P. Sourdaine, S. Galopin, G.E. SeÂralini,
[31] E.A. Thompson, P.K. Siiteri, Utilization of oxygen and Design and synthesis of a new type of nonsteroidal human aroma-
reduced nicotinamide adenine dinucleotide phosphate by tase inhibitors, Bioorg. Med. Chem. Lett. 8 (1998) 1041±1044.
human placental microsomes during aromatization of androste- [48] R.W. Hartmann, H. Bayer, G. Grun, Aromatase inhibitors.
nedione, J. Biol. Chem. 249 (1974) 5364±5372. Synthesis and structure±activity studies of novel pyridyl-substi-
[32] E.A. Thompson, P.K. Siiteri, The involvement of human pla- tuted indanones, indans, and tetralins, J. Med. Chem. 37
cental microsomal cytochrome P-450 in aromatization, J. Biol. (1994) 1275±1281.
Chem. 249 (1974) 5373±5378. [49] M.M. Bradford, A rapid and sensitive method for the quanti-
[33] C.J. Corbin, S. Graham-Lorence, M. McPhaul, J.I. Mason, tation of microgram quantities of protein utilizing the principle
C.R. Mendelson, E.R. Simpson, Isolation of a full-length of protein±dye binding, Anal. Biochem. 72 (1976) 248±254.
cDNA insert encoding human aromatase system cytochrome [50] O. Boussif, F. Lezoualc'h, M.A. Zanta, M.D. Mergny, D.
P450 and its expression in nonsteroidogenic cells, Proc. Natl. Scherman, B. Demeneix, J.P. Behr, A versatile vector for gene
Acad. Sci. USA 85 (1988) 8948±8952. and oligonucleotide transfer into cells in culture and in vivo:
[34] S. Graham-Lorence, B. Amarneh, R.E. White, J.A. Peterson, polyethylenimine, Proc. Natl. Acad. Sci. USA 92 (1995) 7297±
E.R. Simpson, A three-dimensional model of aromatase cyto- 7301.
chrome P450, Protein Sci. 4 (1995) 1065±1080. [51] D.J. Zhou, D. Pompon, S.A. Chen, Stable expression of
[35] E.R. Simpson, Y. Zhao, V.R. Agarwal, M.D. Michael, S.E. human aromatase complementary DNA in mammalian cells: a
Bulun, M.M. Hinshelwood, S. Graham-Lorence, T. Sun, C.R. useful sytem for aromatase inhibitor screening, Cancer Res. 50
Fisher, K. Qin, C.R. Mendelson, Aromatase expression in (1990) 6949±6954.
health and disease, Recent Prog. Horm. Res. 52 (1997) 185± [52] F. Denizot, R. Lang, Rapid colorimetric assay for cell growth
213. and survival. Modi®cations to the tetrazolium dye procedure
[36] J.L. Gaillard, P. Silberzahn, Aromatization of 19-norandrogens giving improved sensitivity and reliability, J. Immunol.
by equine testicular microsomes, J. Biol. Chem. 262 (1987) Methods 89 (1986) 271±277.
5717±5722. [53] P. Dallemagne, P. Sonnet, C. Enguehard, S. Rault, A con-
[37] T. Dintinger, J.L. Gaillard, I. Zwain, R. Bouhamidi, P. venient route to new phenyltetrahydroindolizines, J.
Silberzahn, Synthesis and aromatization of 19-norandrogens in Heterocyclic Chem. 33 (1996) 1689±1694.
the stallion testis, J. Steroid Biochem. 32 (1989) 537±544. [54] R.W. Brueggemeier, N.E. Katlic, E€ects of the aromatase in-
[38] J.L. Gaillard, Equine testicular aromatase: substrates speci®city hibitor 7a-(4'-amino)phenylthio-4-androstene-3,17-dione in
 P. Auvray et al. / Journal of Steroid Biochemistry and Molecular Biology 70 (1999) 59±71 71

MCF-7 human mammary carcinoma cell culture, Cancer Res. [58] J.N. Wright, G. Slatcher, M. Akhtar, `Slow-binding' sixth-
47 (1987) 4548±4551. ligand inhibitors of cytochrome P-450 aromatase: studies with
[55] J. Carmichael, W.G. DeGra€, A.F. Gazdar, J.D. Minna, J.B. 19-thiomethyl- and 19-azido-androstenedione, Biochem. J. 273
Mitchell, Evaluation of a tetrazolium-based semiautomated (1991) 533±539.
colorimetric assay: assessment of chemosensitivity testing, [59] A.D.N. Vaz, M.J. Coon, H. Peegel, K.M.J. Menon,
Cancer Res. 47 (1987) 936±942. Substituted pyridines: nonsteroidal inhibitors of human placen-
[56] J.B. Schenkman, Studies on the nature of the type I and type tal aromatase cytochrome P-450, Drug Metab. Dispos. 20
II spectral changes in liver microsomes, Biochem. 9 (1970) (1992) 108±111.
2081±2091. [60] J.A. Cook, J.B. Mitchell, Viability measurements in mamma-
[57] J.A. Canick, K.J. Ryan, Cytochrome P-450 and the aromatiza- lian cell systems, Anal. Biochem. 179 (1989) 1±7.
tion of 16 alpha-hydroxytestosterone and androstenedione by [61] T. Mosmann, Rapid colorimetric assay for cellular growth and
human placental microsomes, Mol. Cell. Endocr. 6 (1976) 105± survival: application to proliferation and cytotoxicity assays, J.
115. Immun. Methods 65 (1983) 55±63.