Aromatase inhibition by 7-substituted steroids in human choriocarcinoma cell culture

J. Steroid Biochem. Molec. BioL Vol. 41, No. 1, pp. 85-90, 1992 Printed in Great Britain. All rights reserved

AROMATASE IN HUMAN

INHIBITION

0960-0760/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press plc

BY 7-SUBSTITUTED

CHORIOCARCINOMA

CELL

STEROIDS

CULTURE*

ROBERT W. BRUEGGEMEIER,~" NANCY E. KATLIC, CHARLOTTE A. KENREIGH a n d PuI-KAI LI College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210, U.S.A.

(Received 24 October 1990; received for publication 28 August 1991)

Surmnary--Androstenedione analogs containing 7ct-substituents have proven to be potent inhibitors of aromatase both in vitro and in vivo. Several of these agents have exhibited higher affinity for the enzyme complex than the substrate. In order to examine further the interaction(s) of 7-substituted steroids with aromatase, 7-substituted 4,6-androstadiene-3,17diones were synthesized and demonstrated competitive inhibition of aromatase activity in human placental microsomes. 7-Substituted 1,4,6-androstatriene-3,17-diones demonstrated mechanism-based inhibition of placental aromatase activity. These agents were evaluated for inhibition of aromatase activity in the JAr human choriocarcinoma line. The 7-substituted 4,6-androstadiene-3,17-diones produced dose dependent inhibition of aromatase activity in the cell cultures, with IC50values ranging from 490 nM to 4.5/tM. However, these agents are less effective when compared to other steroidal inhibitors, such as 7ct-thiosubstituted androstenediones. These results on the 7-substituted 4,6-androstadiene-3,17-diones are consistent with the data from biochemical enzyme inhibition studies using human placental aromatase. On the other hand, 7-phenethyl-l,4,6-androstatriene-3,17-dione exhibits greater inhibitory activity, with an IC50value of 80 nM. Other mechanism-based inhibitors, 7~t-(4'-amino)phenylthio-1,4androstadiene-3,17-dione and 4-hydroxyandrostenedione, also exhibited potent inhibition of aromatase activity in JAr cells. In summary, the most effective B-ring modified steroidal aromatase inhibitors are those derivatives that can project the 7-aryl substituent into the 7~t-position.

inactivated aromatase by an enzyme-catalyzed process[l 1-13]. Introduction of a 7*t-substituent on 1,4-androstadiene-3,17-dione yielded a potent mechanism-based irreversible inhibitor of aromatase, 7~-(4'-amino)phenylthio-l,4-androstadiene-3,17-dione (7~-APTADD, 2, Fig. 1) [6]. The introduction of substituents at C7 of 4,6-androstadiene-3,17-dione provided analogs (compounds 3-8, Fig. 1) with moderate to good aromatase inhibition in microsomal assays [14]. In addition, introduction of a 1,2-double bond yielded 7-substituted 1,4,6-androstatriene-3,17diones (compound 9, Fig. 1), which demonstrate mechanism-based irreversible inhibition [15]. Current research activities by our laboratory have focused on the biochemical and pharmacological evaluations of 7-substituted 4,6-androstadiene-3,17-diones and 1,4,6-androstatriene-3,17-diones. This manuscript reports the evaluation of aromatase inhibition by these agents (compounds 3 - 9 ) in the JAr human choriocarcinoma line. This cell line has high levels of aromatase activity [16--18] and has been used in evaluation of aromatase inhibitors [19, 20]. In these studies, smaller numbers

INTRODUCTION

Several 7~t-thiosubstituted derivatives of androstenedione have demonstrated enhanced affinity for aromatase and produced very effective inhibition of aromatase activity present in human placental microsomes [1-7]. This group of inhibitors includes competitive, affinity, photoaffinity, and enzyme-activated irreversible inhibitors. One of the most potent competitive inhibitors is 7~-(4'-amino)phenylthio-4-androstene-3,17-dione, 7c~-APTA (1, Fig. 1), with an apparent Kt of 18 nM [1]. Also, it has demonstrated effectiveness in inhibiting aromatase in cell cultures [7, 8] and in treating hormone-dependent rat mammary tumors [7, 9]. Androstenedione derivatives with extended linear conjugation in ring A and/or B produced effective inhibition of aromatase [10]. Furthermore, the introduction of an additional double bond in the A ring resulted in inhibitors that *Portions of this work were presented at the 72nd Annual Meeting of the Endocrine Society, Atlanta, GA, June 20-23 (1990), tTo whom correspondence should be addressed.

85

86

ROBERTW. BRUEGGEMEIERet al. 70t-Substituted androstenedione and androstadienedione

O

O

O

~

%

*

s- - - ~

NH2

7~-APTA 1

7~-APTADD 2

7-Substituted 4,6-androstadienediones

n

O

O

n

R

R

3 4 5

7-benzyl-ADD 7-aminobenzyl-ADD 7-nitrobenzyl-ADD

1 1 1

-H -NH 2 -NO 2

6 7 8

7-phenethyl-ADD 7-aminophenethyl-ADD 7-nitrophenethyl-ADD

2 2 2

-H -NH 2 -NO 2

9

7-phenethyl-ATD

2

-H

7-Substitttted 1,4,6-androstatrienedione O

~ O

(

C

H

2

)~ n

R

Fig. 1. 7-Substituted steroidal aromatase inhibitors.

of cells, less media, and shorter incubation times in measuring enzymatic activity are used when compared with other cell lines [20]. MATERIALS AND METHODS

Commercial steroids were obtained from Steraloids (Wilton, NH) and checked for purity by melting point and thin layer chromatography. 7-Substituted 4,6-androstadiene-3,17diones and 1,4,6-androstatriene-3,17-diones were prepared as previously described [14, 15]. [ 1fl-3H]-4-Androstene-3,17-dione was purchased from New England Nuclear (Boston, MA) and purity checked by TLC. JAr human choriocarcinoma cells were obtained from American Tissue Cell Culture (Bethesda, MD). RPMI media was obtained in powdered form from GIBCO (Long Island, NY). The sterilized liquid media was prepared by the Ohio State University Cell Culture Service, OSU Compre-

hensive Cancer Center, by dissolving the powder in water containing sodium chloride (0.487 g/l), pyruvic acid (0.11 g/l), sodium bicarbonate (1.5 g/l) and phenol red (0.01%) and the pH adjusted to 6.8. Fetal calf serum was obtained from KC Biological (Lenexa, KS). Steroids were removed from the fetal calf serum by two treatments with dextran-coated charcoal [21]. Tissue culture flasks and supplies were obtained from Corning Glass Works (Corning, NY). Biochemicals were purchased from Sigma Chemical Co. (St Louis, MO). Radioactive samples were detected with a Beckman LS 6800 scintillation counter using Formula 963 (New England Nuclear) as the counting solution. IC50 values represent the concentration of inhibitor to produce a half-maximal inhibition of aromatase activity in the JAr cell cultures and were calculated by a nonlinear regression analysis using the Marquardt method (SAS Institute, Cary, NC).

Steroidal aromatase inhibitors in JAr cells

Inhibition of JAr aromatase activity JAr cells were grown in 75 cm 2 plastic flasks at 37°C in RPMI media (10 ml) containing 10% fetal calf serum and gentamycin (20 mg/l). For aromatase activity, JAr cells were divided into 9.4 cm 2 wells in RPMI media (2 ml) containing 10% steroid-free fetal calf serum and gentamycin (20 mg/1). When cultures reached 90% confluency (approximately 3 to 4 x 106cells), media was changed and varying concentrations of aromatase inhibitors (10 -~2 to 10 -6 M in 5/d 95% ethanol) were added. Each experiment was carried out in triplicate, and inhibitors were evaluated in experiments performed at least three different times. Aromatase activity was determined by measuring the conversion of [ 1fl-3H]androstenedione to 3 H 2 0 [ 19, 20]. For aromatase inhibition, [lfl-3H]andro stenedione (50 nM, 2.5 pCi, in 5 #l EtOH) was added to the cultures at the same time as the inhibitor. Control cultures received [lfl3H]androstenedione, 95% ethanol, and no inhibitor. Blank samples contained [lfl3H]androstenedione in media only (no cells). After 2 h the plates were removed from the incubator and the media transferred into 35 ml centrifuge tubes. Chloroform (10 ml) was added to the media, the samples vortexed, incubated at room temperature for 30 min, then centrifuged at 500g for 10min. Three aliquots (500/~l) of the supernant were transferred into 12 x 75 mm culture tubes and 1% dextran coated charcoal solution was added (500/~1). After vortexing, the tubes were incubated at 4°C for 15 min, then

87

centrifuged for 10 min at 2600g. The supernatent was mixed with Formula 963 cocktail and counted by LSC. DNA content of the cultures was determined by the diphenylamine assay [20]. The percent inhibition was determined by dividing the total amount of 3H20 formed (pmol//~g DNA) in the inhibited sample by the amount of 3H20 formed in the uninhibited (control) samples. RESULTS AND DISCUSSION

The eflicacies of 7-substituted 4,6-androstadiene-3,17-diones to inhibit aromatase activity were evaluated in JAr trophoblastic choricarcinoma cells. Aromatase activity was determined by measuring the conversion of [lfl-3H]androstendione to 3H20[19,20 ]. The level of aromatase activity present in the JAr cell cultures was 6.31 (+ 2.17) pmol product formed per 106 cells per hour, or approximately 44.15 pmol per 9.2 cm: well in 2 h. The 7-substituted 4,6-androstadiene-3,17diones exhibited ICs0s ranging from 490 nM to 4.5/~M (Table 1, Figs 2 and 3). These 7-substituted 4,6-androstadiene-3,17-dione analogs, which demonstrated moderate to weak aromatase inhibition in placental microsomal assays [14], exhibited similar activities for aromatase inhibition in the JAr cell cultures. The most effective agent of this group of compounds is 7-(4'-amino)benzyl-4,6-androstadiene-3,17-dione (7-aminobenzyl-ADD, 4), with an IC50 of 490 nM (Fig. 2). The unsubstituted

Table 1. Aromatase inhibition In JAr cells Compound number

Compound name

IC~oa

Log IC~0" Log SE'

Microsomal app. K~c

3 4 5

7-Benzyl-4,6-androstadiene -3,17-dioaes 7-Benzyl-ADD 600riM -6.22 0.357 7-Aminobenzyl-ADD 490nM -6.31 0.141 7-Nitrobenzyl-ADD 928 nM - 6.03 0.141

61 nM 88riM 94 nM

6 7 8

~Phenethyl-<6-androstadiene-~l~diones 7-PhenethyI-ADD 660nM -6.18 0.275 7-Aminophenethyl-ADD 1250riM -5.90 0.659 7-Nitrophenethyl-ADD 4500nM b ---

174nM 88 nM 95 nM

9

7-Phenethyl- 1,4,6-androstatriene- 3,17-dione 7-Phenethyl-ATD 80 nM - 7.097 0.142

172 nM

a ICso values represent the concentration of inhibitor to produce a half-maximal inhibition of aromatase activity in the JAr cell cultures. The IC~0 values were derived from log IC~0 values calculated by a nonlinear regression analysis using the Marquardt method (SAS Institute, Cary, NC). Log SE values were also obtained from this analysis. bDue to the low inhibitory activity and poor solubility of this compound, a complete dose-response curve was not obtained. This IC~0 value was approximated from the dose-response curve; the regression analysis program was not able to calculate precise log ICs0 and log SE values. CApparent K~ values for aromatase inhibition in human placental microsomes were taken from Refs [14] and [15]. SB 41:I-G

ROBERT W. BRUEGGEMEIERet al.

88

100

~" ....

100-

A g 0~

~

6O

0

_,0=o oo 20

0

~ -c o O

40 20

o 7-benzyI-ADD [] 7-arni n oben.z,yl:ADD & 7-nitrobenzyI-ADD

0

I

-4 2

-4 0

~, ,, ~k ~,

I

I ~1~

-8

-8

0 -12 I

-4

Fig. 2. Inhibition of aromatase activity in JAr cell cultures by 7-benzyl-4,6-androstadiene-3,17-diones. JAr cells in 9.4 crn2 wells were incubated for 2 h at 37°C with radiolabeled substrate (30nM) and 7-benzyl-ADD (©), 7aminobenzyl-ADD (I-q), or 7-nitrobenzyl-ADD (A) at concentrations ranging from 100 pM to 10/~M. Aromatase activity was determined by measuring the amount of 3H20 released. Each point represents the average of three determinations, with variation of less than 10%. The value for 100% estradiol formation in control cultures (no inhibitor) was 6.31 (+2.17) pmol product formed per 106 cells per hour, or 44.15 pmol per well in 2 h.

analog, 7-benzyl-4,6-androstadiene-3,17-dione (7-benzyl-ADD, 3), was also an effective inhibitor in the JAr cells. However, substitution of the large nitro moiety on the aromatic ring (7-nitrobenzyl-ADD, 5) lowered activity by a factor of 2. In the 7-phenethyl-4,6-androstadiene-3,17dione series (Fig. 3), the unsubstituted compound 7-phenethyl-ADD (6) was the most active with an IC50 of 660 nM. In contrast to the 100

80 -

"-g-'~%"

R,

"~,\~\

•,~ 40 -- o 20

o - 2

7-phenethyI-ADD \'a [] 7-aminophenethyI-ADD \ \ • • 7-nitrophenethyI-ADD ~,

I -~ o

I

-8

I -s

• 7~-APTA • 7a-APTADD u 4-OH-A

\ \ k\,

0

log [11

"~ •-, c 0 O

80 _

e~e.,._ ~..::.lp "~e~-I~.,~•

J -.

log Ill Fig. 3. Inhibition of aromatase activity in JAr cell cultures by 7-phenethyl-4,6-androstadiene-3,17-diones. JAr cells in 9.4 cm ~ wells were incubated for 2 h at 37°C with radiolabeled substrate (30 nM) and 7-phenethyl-ADD [O], 7aminophenethyl-ADD (r-q), or 7-nitrophenethyl-ADD (ll) at concentrations ranging from 1 nM to 10 #M.

I -10

I -8

g'g-o- !~.=_ I -6 -4

log [q Fig. 4. Inhibition of aromatase activity in JAr cell cultures by 7~t-APTA, 7ct-APTADD, and 4-OH-A. JAr cells in 9.4 cm 2 wells were incubated for 2 h at 37°C with radiolabeled substrate (30 nM) and 7~t-APTADD (ll), 7~-APTA (Q), or 4-OHA (I-q) at concentrations ranging from 10 pM to 10 #M.

7-benzyl series, substitution of an amino functionality at the 4'-position of the aromatic ring (7-aminophenethyl-ADD, 7) slightly reduced aromatase inhibitory activity in the JAr cells. The introduction of the nitro functionality (7nitrophenethyl-ADD, 8) greatly decreased aromatase activity. For comparison, 7~-APTA, 7~-APTADD, and 4-hydroxyandrostenedione (4-OH-A) inhibited aromatase activity in JAr cells in dosedependent fashions (Fig. 4), with ICs0s of 105 nM, 7.3 nM and 3.5 nM, respectively [20]. In microsomal assays, the two 7a-substituted aromatase inhibitors exhibit similar apparent Kts and thus similar affinity for the enzyme complex, while the apparent Ki for 4-OH-A is slightly higher. Interestingly, the inhibition in the JAr cultures by 7~-APTA, a competitive inhibitor, was approx. 10 times less active than 7~-APTADD and 4-OH-A, both mechanismbased inhibitors. Introduction of a 1,2-double bond in the A ring of compound 6 yielded 7-phenethyl-l,4,6androstatriene-3,17-dione (7-phenethyl-ATD, 9), which demonstrated mechanism-based irreversible inhibition in placental microsomal assays [15]. In the JAr cell cultures, this compound was very effective in inhibiting aromatase activity and exhibited and IC50 of 80 nM (Fig. 5). Thus, the addition of the 1,2-double bond led to an increase in inhibitory activity by a factor of 8 compared to 7-phenethyl-ADD, 6. In summary, the introduction of bulky, aryl substitutions at the C-7 position of the B-ring of androstenedione have provided several effective aromatase inhibitors. In JAr cell cultures, 7-substituted 4,6-androstadiene-3,17-diones, corn-

Steroidal aromatase inhibitors in JAr cells • 7a-APTADD a 7 -phenethyI-ATD

~.

1oo o

IE

60

o

o °

o

0

\

~\

40

%

20-

0

-12

I

-10

I

-8

~

-

I

-6

-4

log [I]

Fig. 5. Inhibition of aromatase activity in JAr cell cultures by 7-phenethyl-l,4,6-androstadiene-3,17-dione. JAr cells in 9.4 cm 2 wells were incubated for 2 h at 37°C with radiolabeled substrate (30 nM) and 7-phenethyl-ATD ([]) or 7~t-APTADD (,) at concentrations ranging from 10 pM to 10FM.

pounds 3-8, produce moderate to weak inhibition of aromatase activity. The ICb0 values for JAr aromatase inhibition by these competitive inhibitors range from 490 nM to 4.5 #M, with the amino derivative 4 being the most potent of the series. In contrast, the differences among the six inhibitors were significantly smaller in the human placental microsomal assays, with apparent Ki values ranging from 61 to 174 nM [14]. Also, no differences in apparent K, values were observed among the amino and nitro analogs (compounds 4, 5, 7and 8). Thus, a greater range for aromatase inhibition by these compounds exists in the JAr cells, and differences in potency among specific inhibitors are observed in the cell cultures which were not seen in placental microsomal assays. These results suggest that other parameters, such as lipophilicity, membrane permeability, and nonspecific protein binding, will influence inhibitory activity in intact cells. Mechanism-based inhibitors, also termed enzyme-activated irreversible inhibitors, demonstrated enhanced inhibitory activity of JAr aromatase. The IC50 value for 7-phenethylATD, 9, is 80 nM; similarly, enhanced inhibitory activity in JAr cells were observed for 7~t-APTADD and 4-OH-A, both mechanismbased inhibitors [20]. Furthermore, the doseresponse curve for 7-phenethyl-ATD in the JAr cells was steep and parallel to the dose-response curves for 7ct-APTADD and 4-OH-A. Thus, the effectiveness of mechanism-based aromatase inhibitors observed in the JAr cells is greater than that observed for competitive inhibitors.

89

These results of the inhibition of aromatase activity in JAr cells by various 7-substituted steroidal inhibitors confirm and extend structure-activity relationships on these agents. Those derivatives that can project the 7-aryl substituent into the 7~ pocket are effective inhibitors. However, these inhibitors are not as effective as previously reported 7g-thiosubstituted analogs [6, 8, 20]. Nevertheless, the 7-substituted 4,6-androstadienediones and 7substituted 1,4,6-androstatrienedione may offer an advantage of greater metabolic stability in vivo. Overall, the most effective B-ring modified aromatase inhibitors are those derivatives with aryl substitution at the 7~-position. These resuits suggest that additional interactions occur between the phenyl ring at the 7at-position and amino acids at or near the enzymatic site of aromatase to result in enhanced affinity of the inhibitors. Acknowledgements--This research work was supported in part by NIH Grant RO1 DK-40255 (R.W.B.), American Cancer Society Grant BC-482 (R.W.B.), and NIH Grant P30-CA16058 (OSU Comprehensive Cancer Center).

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