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Imidazole derivatives of pyrrolidonic and piperidonic acids as potential inhibitors of human placental aromatase in vitro
J. Steroid Biochem. Molec. Biol. Vol. 57, No. 1/2, pp. 73-77, 1996 Copyright © 1996 Elsevier Science Ltd 0960-0760(95)00253-7 Printed in Great Britain. All rights reserved 0960-0760/96 $15.00 + 0.00
Pergamon
I m i d a z o l e D e r i v a t i v e s of P y r r o l i d o n i c and P i p e r i d o n i c A c i d s as P o t e n t i a l I n h i b i t o r s of H u m a n P l a c e n t a l A r o m a t a s e In V i t r o M o o m e n Baroudi, Jacqueline Robert and Cuong Luu-Duc* Laboratoire de Chimie-Pharmacie, Unit~ de Recherche Associ~e au C N R S no 1287, Universitd ffoseph-Fourier, Grenoble I, F 38706 La Tronche Cedex, France
Inhibitory activities towards human placental aromatase of novel pyrrolidinone and piperidinone d r u g s w e r e i n v e s t i g a t e d a n d c o m p a r e d w i t h t h o s e o f a m i n o g l u t e t h i m i d e ( A G ) in vitro. All c o m p o u n d s s h o w i n g a s t r o n g e r i n h i b i t o r y e f f e c t t h a n this o f A G h a d t h e f o l l o w i n g c o m m o n s t r u c t u r a l f e a t u r e : a n i m i d a z o l e s i d e - c h a i n in C-3 p o s i t i o n , w i t h a s u b s t i t u t e d o r n o n - s u b s t i t u t e d a r o m a t i c r i n g in t h e C-2 p o s i t i o n a n d a n a l i p h a t i c c h a i n ( n - b u t y l o r n - o c t y l ) o r a p h e n y l m o i e t y o n t h e n i t r o g e n o f t h e p y r r o l i d o n e o r p i p e r i d o n e r i n g . W h e n t h e C-3 s i d e - c h a i n d i d n o t b e a r a n y i m i d a z o l e r i n g , n o a c t i v i t y was o b s e r v e d . R e s p e c t i v e K i v a l u e s f o r t h e c o m p e t i t i v e i n h i b i t i o n e x e r t e d b y t h e m o r e p o t e n t i n h i b i t o r s 10, 11, 13 a n d 21 w i t h a n d r o s t e n e d i o n e as s u b s t r a t e w e r e 19.2, 20.3, 16.8 a n d 1 5 . 4 p M , r e s p e c t i v e l y (Ki A G := 77.0/~M). C o p y r i g h t © 1996 E l s e v i e r S c i e n c e L t d .
J. Steroid Biochem. Molec. Biol., Vol. 57, No. 1/2, pp. 73-77, 1996
INTRODUCTION
heteroatom. Most of them exhibit competitive inhibition through binding of this heteroatom to the heine iron of cytochrome P450 enzyme. T h e inhibition is maximum for compounds bearing aromatic substituents [7, 8] which probably occupy a lipophilic region of the apoprotein moiety of the P450. Imidazole or triazole compounds such as fadrozole [4-(5,6,7,8-tetrahydroimidazo[1,5a] pyridine-5-yl) benzonitrile, HC1] (CGS 16949A); letrozole (4-[1(cyanophenyl)-l-(1,2,4-triazolyl)-methyl] benzo nitrile) (CGS 20267) [9] and (3ctR)-trans-l-[(3ct-ethyl9-(ethylthio)-2,3,3~,4,5,6-hexahydro- 1H-phenalen-2yl)methyl]-lH-imidazole, HCI) (Org 33201) [10] are new effective azole-type aromatase inhibitors and some of them are in clinical trial phase. Antifungal derivatives such as thioconazole, econazole or miconazole are potent inhibitors of aromatase [7, 11]. We have previously reported on the synthesis and in vitro aromatase inhibition of imidazole and benzimidazole derivatives [ 12], most of them are more potent than A G in vitro. T h e aim of the present study was to determine the aromatase inhibition activity of a series of pyrrolidonic or piperidonic acid new compounds. T h e pyrrolidonic and piperidonic structures are related to the tobacco alkaloid derivatives (cotinine, nicotine, anabasine) whose aromatase inhibition activity has been investigated [13-16].
Estrogens are believed to play a key role in the development and growth of breast tumours in postmenopausal women, so many forms of therapy have been directed towards reducing estrogen stimulation of t u m o u r cell proliferation [1]. Aromatase, a cytochrome P450 enzyme, catalyses the bioconversion of androgens to estrogens through the aromatization of the A ring of androgens, so inhibition of aromatase has been identified as a good therapeutic strategy for the treatment of estrogen-dependent breast cancer through the regulation of estrogen biosynthesis. Various steroidal and non-steroidal inhibitors have been developed and well reviewed [2-6]. Aminoglutethimide (AG) is presently the only non-steroidal aromatase inhibitor co~a~mercially available, but it also inhibits enzymes involved in adrenal steroidogenesis at similar concentrations. This lack of specificity and toxicity side effects ,;purred the recent interest in development of safer and more selective inhibitors. Some of them are based on the imidazole structure. This class of compounds contains a suitable positioned
*Correspondence to C. LulL-Due. Received 24 Mar. 1995; accepted 18 Sep. 1995. 73
74
Moomen Baroudi et al.
Table 1. Structure and K i values of the tested compounds (K i A C = 77.0 ~tM)
"r? o R / Compound
(I)n
n
Ar
R1
R2
1 2
2 2
4 - N H 2 C6H 4 2,4-C12 C6H 3
n-C4H 9 "
3 4
2 2
C6H 5 "
C6H 5 n-Call 9
5 6
2 2
4 - N O 2 C6H 4 4-C1 C6H 4
COOH
--
CO-Im
64.5
"
59.6 32.4
"
.
.
.
7
2
4-F C6H4
8 9
2 2
4 - N H z C6H 4 4-CsH4N
.
48.6
"
10
2
2,4-C12 C6H 3
.
11 12 13
2 2 2
4 - C N C6H 4 4-C1 C6H 4 2,4-C1 z C6H 3
14 15 16
2 1 1
4-C1 C6H 4 4 - F C6H 4 C6H 5
C6H 5 rt-eaH 9 "
17 18 19
2 1 2
C6H 5 4 - F C6H 4 4-C1 C6H 4
.
.
.
.
.
.
.
.
20 21
2 1
C6H 5 4 - F C6H 4
.
CH2-O-CO-Im . .
"
49.3 52.7
.
.
19.2
. . . n-C8H17 "
.
20.3 25.9 16.8
.
.
K i (#M)
"
" Benzimidazole
31.7 45.5 ---28.9 15.4
EXPERIMENTAL Chemicals
T h e tested compounds were synthetized and identified as described previously [17]. T h e i r chemical structures are shown in T a b l e 1. [1,2,6,7 3H]androst-4ene-3,17-dione ( 9 8 C i / m m o l ) was purchased from Amersham, d,l-aminoglutethimide from Ciba-Geigy, 4-androstene-3,17-dione and N A D P H from Sigma Chemical. Radioactivity was measured on a Packard liquid scintillation spectrophotometer.
bation tube contained labeled and unlabeled androstenedione in buffer at varying concentrations ranging from 15 to 100 n M in an ethanolic solution, with and without putative inhibitors. T h e solvent was evaporated under nitrogen and 0.5 ml of 10 m M phosphate buffer (pH = 7.5) with E D T A 1 m M , KC1 100 m M and one drop of propylene glycol was added to the residue. T h e reaction was initiated with 100 #1 of the microsomal suspension (0.14mg/ml) with N A D P H 0.5 m M . T h e incubations were carried out at 37°C in a shaking water bath during 30 min. Indeed, for a given concentration of androstenedione of 0.1 # M in microsomal suspension (0.14mg/ml), the graph of the water released as a function of time is linear during the first 20 rain and then flattens out; since the speed varies little afterwards, the parameter incubation time is chosen to 30 min. T h e assays were run in duplicate and samples were removed at 2 min interval. T h e reaction was stopped by the addition of chloroform (Sml) and vortexing 30 s. After centrifugation at 1500 g, 100 pl were removed from the water phase (in duplicate) and mixed with 10ml of the scintillation solution. T h e extent of aromatization was determined by measuring the amount of tritiated water released after aromatization of [1,2,6,7 3H]androst-4-ene-3,17dione as described by Rabe [22]. T h e K m and Vmax] values were determined by the L i n e w e a v e r - B u r k plots. T h e h u m a n placental microsomes preparation used in this study showed an apparent K m for androstenedione of 0.08 p M . T h e inhibitory activity of the tested compounds was determined by 3H20 release from androstenedione as an index of residual aromatase activity. T h e inhibitors were added at various concentrations (0, 20, 5 0 p M ) to the incubation m e d i u m prior to the addition of the microsomes. T h e apparent Ki values were determined by the least square analysis of secondary replots (slope vs [I]) of L i n e w e a v e r - B u r k plots (1/V vs 1/S). T h e points plotted are the mean of the four determinations. R E S U L T S AND D I S C U S S I O N
Arom a t a s e assay
T h e inhibitory activities of the compounds towards aromatase were determined in vitro using h u m a n placental microsomes and [1, 2, 6, 7 ~H]androst-4-ene3,17-dione. T h e microsomal fraction was prepared from h u m a n t e r m placenta obtained immediately after delivery and carried out at - 4 ° C . Microsomes were extracted according to Ryan [18]. After isolation, microsomes were suspended in phosphate buffer 10 m M , K C I 100 m M , E D T A 1 m M , then frozen and stored in aliquots of 0.5 ml at - 70°C. Protein concentration, determined by the method of L o w r y et al. [19], was 10.6mg of protein/ml. T h e methodology for the determination of Ki values has been described previously [18, 20, 21]. Each incu-
T h e derivates are competitive-type inhibitors of aromatase with apparent Ki values of 15-64/~M (Ki = 77.0 p M for AG, the reference compound). T h e results presented in T a b l e 1 show that all new imidazole compounds of the series inhibit aromatase more potently than A G in vitro (see also Figs 1-4). F r o m an initial SAR study of imidazole [12] and piperidone carboxylic acids (unpublished results), and as noticed by Jones et al. [23], we observed that the lack of aryl moities in imidazole series and the lack of suitable nitrogen heterocycle produced compounds that were inactive towards aromatase. T h e most suitable substituents for aryl are 2,4-C1z or 4 - C N moities. T h e presence of an imidazole on the C-3 of the lactame ring is absolutely necessary for inhibitory activity. N o n e of the 3-carboxylic compounds (1, 2, 3) or
Imidazole Derivatives: Inhibitors of Aromatase 0.8 "7,
0.7
el C-'
i!
0.6
c~
0.5
~L
E~
"~
•
0 p.M AG
•
20 ttM AG
•
50 tdVl A G
75
0.4 0.3
0.2 0.1 I
-20
-10
I
I
I
I
I
I
I
I
0
10
20
30
40
50
60
70
-1 /Androstenedione (~M)
Fig. 1. D o u b l e - r e c i p r o c a l
plot s h o w i n g the effect o f s u b s t r a t e ( [ 3 H ] a n d r o s t e n e d i o n e ) c o n c e n t r a t i o n on a r o m a tase r e a c t i o n v e l o c i t y in the p r e s e n c e of 0, 20 and 50/tM (AG).
benzimidazoles (16, 17, 18, 19) were inhibitors, even if a p r i m a r y amine moiety on the aryl ring (1) is usually a positive requirement for inhibitory activity. C o m p o u n d 15, where the piperidone ring is replaced by a pyrrolidone, differs by one methylene group in the lactam ring and proved to be slightly more potent than 7. Variation of the aromatic substituents induced changes in inhibitory activity. As expected from earlier work with aryl benzimidazole and imidazole compounds [7, 12] and other nitrogen heterocycles [23], the greatest inhibitory act:ivity is obtained with electronwithdrawing substituents. T h e order of decreasing inhibitory effect for aryl substitutions is: 2,4-C12 >~4-
0.012
C N > 4-C1 > 4 - F >~ 4 - N H z > 4-CsH4N > 4-NO2 > H. Maximal activity was achieved by replacing two hydrogens by two chloro at the 2,4-positions, or one hydrogen by a C N at the 4-position. O f nearly equal importance to the choice of the aromatic substituents is the way in which the lactam ring is attached to the imidazole. C o m p o u n d s 20 and 21 differed from 4 and 15 by one methylene moiety and they are 3 times more potent. In order to define the effect of varying the substitution on the N - 1 atom of the lactam ring, we synthetized and tested the N - o c t y l 12, 13 and the N - p h e n y l 14, analogs of the N - b u t y l 6 compound; 6 and 14 have about the same activity, but the N octyl substitution enhanced slightly the inhibitory activity.
T
0.010 -[CONCLUSION
iI ~ ~ l -80
-60
I
I 0.002 -40
..20
0 20 A G ($~vl)
40
60
Fig. 2. G r a p h s h o w i n g the effect o f A G c o n c e n t r a t i o n on slope o f the d o u b l e - r e c i p r o c a l plot lines. T h e K i v a l u e ( n e g a t i v e x i n t e r c e p t ) , r e f l e c t i n g i n h i b i t o r effectiveness, w a s 77.0/~M.
T h e new non-steroidal lactam-compounds described here are active towards h u m a n placental aromatase in vitro. T h e y inhibit aromatase more potently than AG, the reference starting point. T h e i r inhibitory properties depend on the type of the imidazole moiety, but also strongly on the position and the type of substituents at the benzene nucleus. In agreement with this structure-activity relationship, a c o m m o n structural feature of the inhibitory lactam drugs was the presence of one substituted aromatic ring on the C-2 and one imidazole on the C-3 of the piperidone or pyrrolidone ring. T h e
76
M o o m e n B a r o u d i et al.
6.0
5.0 v....4
•
0 laM 21
*
20 ~tM 21
•
50 g M 21
.=,.~
4.0 OO @
3.0
4~
2.0 ID
>.
1.0 '
-60
-40
-20
0
20
I 40
I 60
I 80
Androstenedione (pM) -1 Fig. 3. D o u b l e - r e c i p r o c a l plot showing t h e effect of s u b s t r a t e ([3H]androstenedione) c o n c e n t r a t i o n on a r o m a tase r e a c t i o n velocity in t h e p r e s e n c e of 0, 20 a n d 501aM (21).
most aryl suitable substituents are 2,4-Clz or 4-CN m o i e t i e s . T h e g o o d i n h i b i t o r y a c t i v i t y o f 10 a n d 13 l e d us to investigate the influence of the nature and the length of the C-3 side-chain. The introduction of a methylene-oxy moiety between the carbonylimidazole and the lactam ring enhanced the activity significantly.
0.08 0.07 0.06 ~0.05 0.04 0.03 0.02
I---
-20
- 10
0
I
I
I
I
I
10
20
30
40
50
21 ( ~ ) Fig. 4. G r a p h showing t h e effect o f 21 c o n c e n t r a t i o n on slope of t h e d o u b l e - r e c i p r o c a l plot lines. T h e K i value (negative x i n t e r c e p t ) , reflecting i n h i b i t o r effectiveness, was 15.3 #.
Acknowledgements--The authors are grateful to the Laboratoire de Biologie Cellulaire (Dr P. Ravanel) for the extraction of the microsomal fraction by ultracentrifugation.
REFERENCES
1. James V. H. T. and Reed M. J.: Steroid hormones and human cancer. Prog. Cancer Res. Ther. 14 (1980) 471-487. 2. Van Wanve J. P. and Jansen P. A. J.: Is there a case for P-450 inhibitors in cancer treatment? J. Med. Chem. 32 (1989) 2231-2239. 3. Perez N. and Borja J.: Aromatase inhibitors: clinical pharmacology and therapeutic implications in breast cancer. J. Int. Med. Res. 20 (1992) 303-312. 4. Banting L., Nicholls P. J., Shaw M. A. and Smith H. J.: Recent developments in aromatase inhibition as potential treatment for oestrogen-dependent breast cancer. Prog. Med. Chem. 26 (1989) 253-298. 5. Brodie A., Brodie H. B., Callard G., Robinson C., Roselli C. and Santen R.: Recent advances in steroid biochemistry and molecular biology, ft. Steroid Biochem. Molec. Biol. 44 (1993) 321-695. 6. Brodie A. and Santen R.: Aromatase and its inhibitors in breast cancer treatment. Breast Cancer Res. Treat. 30 (1994) 1-115. 7. Ayub M. and Levell M. J.: Structure-activity relationships of the inhibition of human placental aromatase by imidazole drugs including ketoconazole, ft. Steroid Biochem. 31 (1988) 65-72. 8. Nicholls P. J. and Shaw M. A.: Inhibition of human placental aromatase by substituted azoles. Br. ft. Pharmac. 92 (1987) 726P. 9. Lang M., Batzl Ch., Furet P., Bowman R., H/iussler A. and Bhamagar A. S.: Structure-activity relationships and binding model of novel aromatase inhibitors, ft. Steroid Biochem. Molec. Biol. 44 (1993) 421-428.
Imidazole Derivatives: I n h i b i t o r s o f Aromatase 10. Geelen J. A. A., Loozen H. J. J., Deckers G. H., de Leeuw R., Kloosterboer H. J. and Lamberts S. W. J.: ORG. 33201: A new highly selective orally ~lctive aromatase inhibitor. J. Steroid Biochem. Molec. Biol. 44 (1993) 681-682. 11. Mason J. I., Carr B. R. and Murray B. A.: Imidazole animycotics: selective inhibitors of steroid aromatization and progesterone hydroxylation. Steroids 50 (1987) 179-189. 12. Hida F., Robert J. and L au-Duc C.: Synthesis and evaluation of benzimidazole and imidazole compounds as potential aromatase inhibitors. Farmaco 49 Ct994) 489--492. 13. Barbieri R. L., Gochberg J. and Ryan K. J.: Nicotine, cotinine and anabasine inhibit aromatase in human trophoblast in vitro. J. Clin. Invest. 77 (1986) 1727-1733. 14. Osawa Y., Tochigi B., q[~ochigi M., Ohnishi S., Watanabe Y., Bullion K., Osawa G., Nakabayashi Y. and Yarborough C.: Aromatase inhibitors in c:igarette smoke, tobacco leaves and other plants. J. Enzyme Inhib. 4 (1990) 187-200. 15. Bullion K., Ohnishi S. and Osawa Y.: Competitive inhibition of human placental arornatase by N-n-octanoylnornicotine and other nornicotine derivatives. Endocrine Res. 17 (1991) 409--419. 16. Kadohama N., Shintani K. and Osawa Y.: Tobacco alkaloid derivatives as inhibitors of breast cancer aromatase. Cancer Lett. 75 (1993) 175-182.
77
17. Baroudi M., Robert J. and Luu-Duc C.: Imidazolides of piperidone carboxylic acids: synthesis and physical properties. Heterocyclic Comm. 1 (1995) 147-151. 18. Ryan K. J.: Biological aromatization of steroids, ft. Biol. Chem. 234 (1959) 268-272. 19. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193 (1951) 265-275. 20. Covey D. F., Hood W. F. and Parikh V. D.: 10 fl-propynyl-substituted steroids. J. Biol. Chem. 256 (1981) 1076-1079. 21. Thompson E. A. Jr. and Siiteri P. K.: Utilization of oxygen and reduced nicotinamide adenine dinucleotide phosphate by human placental microsomes during aromatization of androstenedione. J. Biol. Chem. 249 (1974) 5364-5372. 22. Rabe T., Rabe D. and Runnenbaum B.: New aromatase assay and its application for inhibitory studies of aminoglutethimide on microsomes of human term placenta, ft. Steroid. Biochem. 17 (1982) 305-309. 23. Jones C. D., Winter M. A., Hirsch K. S., Stamm N., Taylor H. M., Holden H. E., Davenport J. D., Krumkalns E. V. and Suhr R. G.: Estrogen synthetase inhibitors. Comparison of the in vitro aromatase inhibitory activity for a variety of nitrogen heterocycles substituted with diarylmethane or diarylmethanol groups. J. Med. Chem. 33 (1990) 416--429.
Pergamon
I m i d a z o l e D e r i v a t i v e s of P y r r o l i d o n i c and P i p e r i d o n i c A c i d s as P o t e n t i a l I n h i b i t o r s of H u m a n P l a c e n t a l A r o m a t a s e In V i t r o M o o m e n Baroudi, Jacqueline Robert and Cuong Luu-Duc* Laboratoire de Chimie-Pharmacie, Unit~ de Recherche Associ~e au C N R S no 1287, Universitd ffoseph-Fourier, Grenoble I, F 38706 La Tronche Cedex, France
Inhibitory activities towards human placental aromatase of novel pyrrolidinone and piperidinone d r u g s w e r e i n v e s t i g a t e d a n d c o m p a r e d w i t h t h o s e o f a m i n o g l u t e t h i m i d e ( A G ) in vitro. All c o m p o u n d s s h o w i n g a s t r o n g e r i n h i b i t o r y e f f e c t t h a n this o f A G h a d t h e f o l l o w i n g c o m m o n s t r u c t u r a l f e a t u r e : a n i m i d a z o l e s i d e - c h a i n in C-3 p o s i t i o n , w i t h a s u b s t i t u t e d o r n o n - s u b s t i t u t e d a r o m a t i c r i n g in t h e C-2 p o s i t i o n a n d a n a l i p h a t i c c h a i n ( n - b u t y l o r n - o c t y l ) o r a p h e n y l m o i e t y o n t h e n i t r o g e n o f t h e p y r r o l i d o n e o r p i p e r i d o n e r i n g . W h e n t h e C-3 s i d e - c h a i n d i d n o t b e a r a n y i m i d a z o l e r i n g , n o a c t i v i t y was o b s e r v e d . R e s p e c t i v e K i v a l u e s f o r t h e c o m p e t i t i v e i n h i b i t i o n e x e r t e d b y t h e m o r e p o t e n t i n h i b i t o r s 10, 11, 13 a n d 21 w i t h a n d r o s t e n e d i o n e as s u b s t r a t e w e r e 19.2, 20.3, 16.8 a n d 1 5 . 4 p M , r e s p e c t i v e l y (Ki A G := 77.0/~M). C o p y r i g h t © 1996 E l s e v i e r S c i e n c e L t d .
J. Steroid Biochem. Molec. Biol., Vol. 57, No. 1/2, pp. 73-77, 1996
INTRODUCTION
heteroatom. Most of them exhibit competitive inhibition through binding of this heteroatom to the heine iron of cytochrome P450 enzyme. T h e inhibition is maximum for compounds bearing aromatic substituents [7, 8] which probably occupy a lipophilic region of the apoprotein moiety of the P450. Imidazole or triazole compounds such as fadrozole [4-(5,6,7,8-tetrahydroimidazo[1,5a] pyridine-5-yl) benzonitrile, HC1] (CGS 16949A); letrozole (4-[1(cyanophenyl)-l-(1,2,4-triazolyl)-methyl] benzo nitrile) (CGS 20267) [9] and (3ctR)-trans-l-[(3ct-ethyl9-(ethylthio)-2,3,3~,4,5,6-hexahydro- 1H-phenalen-2yl)methyl]-lH-imidazole, HCI) (Org 33201) [10] are new effective azole-type aromatase inhibitors and some of them are in clinical trial phase. Antifungal derivatives such as thioconazole, econazole or miconazole are potent inhibitors of aromatase [7, 11]. We have previously reported on the synthesis and in vitro aromatase inhibition of imidazole and benzimidazole derivatives [ 12], most of them are more potent than A G in vitro. T h e aim of the present study was to determine the aromatase inhibition activity of a series of pyrrolidonic or piperidonic acid new compounds. T h e pyrrolidonic and piperidonic structures are related to the tobacco alkaloid derivatives (cotinine, nicotine, anabasine) whose aromatase inhibition activity has been investigated [13-16].
Estrogens are believed to play a key role in the development and growth of breast tumours in postmenopausal women, so many forms of therapy have been directed towards reducing estrogen stimulation of t u m o u r cell proliferation [1]. Aromatase, a cytochrome P450 enzyme, catalyses the bioconversion of androgens to estrogens through the aromatization of the A ring of androgens, so inhibition of aromatase has been identified as a good therapeutic strategy for the treatment of estrogen-dependent breast cancer through the regulation of estrogen biosynthesis. Various steroidal and non-steroidal inhibitors have been developed and well reviewed [2-6]. Aminoglutethimide (AG) is presently the only non-steroidal aromatase inhibitor co~a~mercially available, but it also inhibits enzymes involved in adrenal steroidogenesis at similar concentrations. This lack of specificity and toxicity side effects ,;purred the recent interest in development of safer and more selective inhibitors. Some of them are based on the imidazole structure. This class of compounds contains a suitable positioned
*Correspondence to C. LulL-Due. Received 24 Mar. 1995; accepted 18 Sep. 1995. 73
74
Moomen Baroudi et al.
Table 1. Structure and K i values of the tested compounds (K i A C = 77.0 ~tM)
"r? o R / Compound
(I)n
n
Ar
R1
R2
1 2
2 2
4 - N H 2 C6H 4 2,4-C12 C6H 3
n-C4H 9 "
3 4
2 2
C6H 5 "
C6H 5 n-Call 9
5 6
2 2
4 - N O 2 C6H 4 4-C1 C6H 4
COOH
--
CO-Im
64.5
"
59.6 32.4
"
.
.
.
7
2
4-F C6H4
8 9
2 2
4 - N H z C6H 4 4-CsH4N
.
48.6
"
10
2
2,4-C12 C6H 3
.
11 12 13
2 2 2
4 - C N C6H 4 4-C1 C6H 4 2,4-C1 z C6H 3
14 15 16
2 1 1
4-C1 C6H 4 4 - F C6H 4 C6H 5
C6H 5 rt-eaH 9 "
17 18 19
2 1 2
C6H 5 4 - F C6H 4 4-C1 C6H 4
.
.
.
.
.
.
.
.
20 21
2 1
C6H 5 4 - F C6H 4
.
CH2-O-CO-Im . .
"
49.3 52.7
.
.
19.2
. . . n-C8H17 "
.
20.3 25.9 16.8
.
.
K i (#M)
"
" Benzimidazole
31.7 45.5 ---28.9 15.4
EXPERIMENTAL Chemicals
T h e tested compounds were synthetized and identified as described previously [17]. T h e i r chemical structures are shown in T a b l e 1. [1,2,6,7 3H]androst-4ene-3,17-dione ( 9 8 C i / m m o l ) was purchased from Amersham, d,l-aminoglutethimide from Ciba-Geigy, 4-androstene-3,17-dione and N A D P H from Sigma Chemical. Radioactivity was measured on a Packard liquid scintillation spectrophotometer.
bation tube contained labeled and unlabeled androstenedione in buffer at varying concentrations ranging from 15 to 100 n M in an ethanolic solution, with and without putative inhibitors. T h e solvent was evaporated under nitrogen and 0.5 ml of 10 m M phosphate buffer (pH = 7.5) with E D T A 1 m M , KC1 100 m M and one drop of propylene glycol was added to the residue. T h e reaction was initiated with 100 #1 of the microsomal suspension (0.14mg/ml) with N A D P H 0.5 m M . T h e incubations were carried out at 37°C in a shaking water bath during 30 min. Indeed, for a given concentration of androstenedione of 0.1 # M in microsomal suspension (0.14mg/ml), the graph of the water released as a function of time is linear during the first 20 rain and then flattens out; since the speed varies little afterwards, the parameter incubation time is chosen to 30 min. T h e assays were run in duplicate and samples were removed at 2 min interval. T h e reaction was stopped by the addition of chloroform (Sml) and vortexing 30 s. After centrifugation at 1500 g, 100 pl were removed from the water phase (in duplicate) and mixed with 10ml of the scintillation solution. T h e extent of aromatization was determined by measuring the amount of tritiated water released after aromatization of [1,2,6,7 3H]androst-4-ene-3,17dione as described by Rabe [22]. T h e K m and Vmax] values were determined by the L i n e w e a v e r - B u r k plots. T h e h u m a n placental microsomes preparation used in this study showed an apparent K m for androstenedione of 0.08 p M . T h e inhibitory activity of the tested compounds was determined by 3H20 release from androstenedione as an index of residual aromatase activity. T h e inhibitors were added at various concentrations (0, 20, 5 0 p M ) to the incubation m e d i u m prior to the addition of the microsomes. T h e apparent Ki values were determined by the least square analysis of secondary replots (slope vs [I]) of L i n e w e a v e r - B u r k plots (1/V vs 1/S). T h e points plotted are the mean of the four determinations. R E S U L T S AND D I S C U S S I O N
Arom a t a s e assay
T h e inhibitory activities of the compounds towards aromatase were determined in vitro using h u m a n placental microsomes and [1, 2, 6, 7 ~H]androst-4-ene3,17-dione. T h e microsomal fraction was prepared from h u m a n t e r m placenta obtained immediately after delivery and carried out at - 4 ° C . Microsomes were extracted according to Ryan [18]. After isolation, microsomes were suspended in phosphate buffer 10 m M , K C I 100 m M , E D T A 1 m M , then frozen and stored in aliquots of 0.5 ml at - 70°C. Protein concentration, determined by the method of L o w r y et al. [19], was 10.6mg of protein/ml. T h e methodology for the determination of Ki values has been described previously [18, 20, 21]. Each incu-
T h e derivates are competitive-type inhibitors of aromatase with apparent Ki values of 15-64/~M (Ki = 77.0 p M for AG, the reference compound). T h e results presented in T a b l e 1 show that all new imidazole compounds of the series inhibit aromatase more potently than A G in vitro (see also Figs 1-4). F r o m an initial SAR study of imidazole [12] and piperidone carboxylic acids (unpublished results), and as noticed by Jones et al. [23], we observed that the lack of aryl moities in imidazole series and the lack of suitable nitrogen heterocycle produced compounds that were inactive towards aromatase. T h e most suitable substituents for aryl are 2,4-C1z or 4 - C N moities. T h e presence of an imidazole on the C-3 of the lactame ring is absolutely necessary for inhibitory activity. N o n e of the 3-carboxylic compounds (1, 2, 3) or
Imidazole Derivatives: Inhibitors of Aromatase 0.8 "7,
0.7
el C-'
i!
0.6
c~
0.5
~L
E~
"~
•
0 p.M AG
•
20 ttM AG
•
50 tdVl A G
75
0.4 0.3
0.2 0.1 I
-20
-10
I
I
I
I
I
I
I
I
0
10
20
30
40
50
60
70
-1 /Androstenedione (~M)
Fig. 1. D o u b l e - r e c i p r o c a l
plot s h o w i n g the effect o f s u b s t r a t e ( [ 3 H ] a n d r o s t e n e d i o n e ) c o n c e n t r a t i o n on a r o m a tase r e a c t i o n v e l o c i t y in the p r e s e n c e of 0, 20 and 50/tM (AG).
benzimidazoles (16, 17, 18, 19) were inhibitors, even if a p r i m a r y amine moiety on the aryl ring (1) is usually a positive requirement for inhibitory activity. C o m p o u n d 15, where the piperidone ring is replaced by a pyrrolidone, differs by one methylene group in the lactam ring and proved to be slightly more potent than 7. Variation of the aromatic substituents induced changes in inhibitory activity. As expected from earlier work with aryl benzimidazole and imidazole compounds [7, 12] and other nitrogen heterocycles [23], the greatest inhibitory act:ivity is obtained with electronwithdrawing substituents. T h e order of decreasing inhibitory effect for aryl substitutions is: 2,4-C12 >~4-
0.012
C N > 4-C1 > 4 - F >~ 4 - N H z > 4-CsH4N > 4-NO2 > H. Maximal activity was achieved by replacing two hydrogens by two chloro at the 2,4-positions, or one hydrogen by a C N at the 4-position. O f nearly equal importance to the choice of the aromatic substituents is the way in which the lactam ring is attached to the imidazole. C o m p o u n d s 20 and 21 differed from 4 and 15 by one methylene moiety and they are 3 times more potent. In order to define the effect of varying the substitution on the N - 1 atom of the lactam ring, we synthetized and tested the N - o c t y l 12, 13 and the N - p h e n y l 14, analogs of the N - b u t y l 6 compound; 6 and 14 have about the same activity, but the N octyl substitution enhanced slightly the inhibitory activity.
T
0.010 -[CONCLUSION
iI ~ ~ l -80
-60
I
I 0.002 -40
..20
0 20 A G ($~vl)
40
60
Fig. 2. G r a p h s h o w i n g the effect o f A G c o n c e n t r a t i o n on slope o f the d o u b l e - r e c i p r o c a l plot lines. T h e K i v a l u e ( n e g a t i v e x i n t e r c e p t ) , r e f l e c t i n g i n h i b i t o r effectiveness, w a s 77.0/~M.
T h e new non-steroidal lactam-compounds described here are active towards h u m a n placental aromatase in vitro. T h e y inhibit aromatase more potently than AG, the reference starting point. T h e i r inhibitory properties depend on the type of the imidazole moiety, but also strongly on the position and the type of substituents at the benzene nucleus. In agreement with this structure-activity relationship, a c o m m o n structural feature of the inhibitory lactam drugs was the presence of one substituted aromatic ring on the C-2 and one imidazole on the C-3 of the piperidone or pyrrolidone ring. T h e
76
M o o m e n B a r o u d i et al.
6.0
5.0 v....4
•
0 laM 21
*
20 ~tM 21
•
50 g M 21
.=,.~
4.0 OO @
3.0
4~
2.0 ID
>.
1.0 '
-60
-40
-20
0
20
I 40
I 60
I 80
Androstenedione (pM) -1 Fig. 3. D o u b l e - r e c i p r o c a l plot showing t h e effect of s u b s t r a t e ([3H]androstenedione) c o n c e n t r a t i o n on a r o m a tase r e a c t i o n velocity in t h e p r e s e n c e of 0, 20 a n d 501aM (21).
most aryl suitable substituents are 2,4-Clz or 4-CN m o i e t i e s . T h e g o o d i n h i b i t o r y a c t i v i t y o f 10 a n d 13 l e d us to investigate the influence of the nature and the length of the C-3 side-chain. The introduction of a methylene-oxy moiety between the carbonylimidazole and the lactam ring enhanced the activity significantly.
0.08 0.07 0.06 ~0.05 0.04 0.03 0.02
I---
-20
- 10
0
I
I
I
I
I
10
20
30
40
50
21 ( ~ ) Fig. 4. G r a p h showing t h e effect o f 21 c o n c e n t r a t i o n on slope of t h e d o u b l e - r e c i p r o c a l plot lines. T h e K i value (negative x i n t e r c e p t ) , reflecting i n h i b i t o r effectiveness, was 15.3 #.
Acknowledgements--The authors are grateful to the Laboratoire de Biologie Cellulaire (Dr P. Ravanel) for the extraction of the microsomal fraction by ultracentrifugation.
REFERENCES
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17. Baroudi M., Robert J. and Luu-Duc C.: Imidazolides of piperidone carboxylic acids: synthesis and physical properties. Heterocyclic Comm. 1 (1995) 147-151. 18. Ryan K. J.: Biological aromatization of steroids, ft. Biol. Chem. 234 (1959) 268-272. 19. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193 (1951) 265-275. 20. Covey D. F., Hood W. F. and Parikh V. D.: 10 fl-propynyl-substituted steroids. J. Biol. Chem. 256 (1981) 1076-1079. 21. Thompson E. A. Jr. and Siiteri P. K.: Utilization of oxygen and reduced nicotinamide adenine dinucleotide phosphate by human placental microsomes during aromatization of androstenedione. J. Biol. Chem. 249 (1974) 5364-5372. 22. Rabe T., Rabe D. and Runnenbaum B.: New aromatase assay and its application for inhibitory studies of aminoglutethimide on microsomes of human term placenta, ft. Steroid. Biochem. 17 (1982) 305-309. 23. Jones C. D., Winter M. A., Hirsch K. S., Stamm N., Taylor H. M., Holden H. E., Davenport J. D., Krumkalns E. V. and Suhr R. G.: Estrogen synthetase inhibitors. Comparison of the in vitro aromatase inhibitory activity for a variety of nitrogen heterocycles substituted with diarylmethane or diarylmethanol groups. J. Med. Chem. 33 (1990) 416--429.