enantioselectivity of aromatase inhibitors: substituted 3-(4-aminophenyl)pyrrolidine-2,5-diones

CHIRALITY 737C380 (1995)

Enantioselectivity of Aromatase Inhibitors: Substituted 3-(4-Aminophenyl)pyrrolidine-2,5-diones

CHRISTOPHER PEPPER, H. JOHN SMITH, PAUL J. NICHOLLS, KEVIN J. BARRELL, AND MASSOUD AHMADI

Welsh School of Pharmacy, University of Wales College of Card$ Cardiff; Wales, United Kingdom

ABSTRACT The (+I-, (-)-, and (+)-forms of 1- and 1,3-substituted 3-(4-aminophenyl)pyrrolidine-2,5-dione have been examined as inhibitors of P450AKOM and P45OcScc. The mhlbitory potency for P450AKOM resided in the (+I-enantiomers of (l), (21, and (4) and the (-)-enantiomus of (3) and (5). These findings have been accommodated within a molecular graphics-derived model for binding of P450AKC)M inhibitors to the substrate binding site. Crystallography showed that (+I421 has the (R)-configuration. Spectral binding studies with human placental P450.4KOM showed type I1 binding but although the Ks values were in line with the IC,,, values for individual compounds there was no overall correlation between Ks and IC,, within the series. There was little difference in the inhibitory potency of the enantiomers and racemate of individual compounds toward P45OcScc. 0 1995 Wiley-Liss, Inc.

KEY WORDS: aromatase, P450AKOMf cholesterol side chain cleavage enzyme, P45OcScc, spectral binding studies, inhibition, substituted 3-(4-aminophenyl)pyrrolidine-2,5-diones, aminoglutethimide

Postmenopausal women presenting with breast cancer are frequently administered tamoxifen after surgery and radio- therapy. Tamoxifen is an estrogen receptor antagonist and removes the estrogen stimulus’ to growth of the life-threat- ening, widespread metastases. If a relapse occurs on tamoxifen, estrogen receptor-positive (ER+) women may be administered an aromatase (P450AKC)M) inhibitor. ‘ P450AK()M catalyses the production of estrone and estradiol from androstenedione and testosterone, respectively, and its inhibition leads to decreased plasma estrogen levels, so removing the stimulus to tumour growth.

Aminoglutethimide (AG) is a reversible competitive inhibitor of P450AKC)M and has been used for some time in the clinic.” It has several undesirable side effect^,:'.^ e.g., CNS depression and blood dyscrasias, and lacks specificity toward the target enzyme. A weaker idubitory effect on the cholesterol side chain cleavage enzyme (P45OCscc) interferes with the conversion of cholesterol to pregnenolone leading to de- pletion of corticosteroids so use of AG requires supplementa- tion with hydrocortisone. These undesirable effects of AG have led to the development of more potent and specific inhibitors of P450AK(]M without CNS effects, i.e., fadrazole,, vorozole.6 We have described a series of N-alkylated 344- aminophenyl) pyrrolidine-2,5-diones, 73 e. g., N-pentyl (3), which fulfill these requirements for a “clean” inhibitor.

P450AKOM inhibitors exhibit stereospecificity to the target enzyme. In AG9 and rogletimide,“’ the pyridine analogue, inhibitory activity residues in the (+)-(R)-enantiomer. We have examined a series of 1- and 1,3-substituted 344-aminophenyl)pyrrolidine-2,5-diones (2)-(5) for enantioselectiv-

ity as inhibitors of P450AK<]M using spectral binding and enzyme inhibition studies.

MATERIALS AND METHODS Materials

1)- and L-Tartaric acid and sodium carbonate were obtained from Aidrich Chemical Company (Poole). Analar grade methanol, ethanol, methylene chloride, and diethyl ether were obtained from Fisons Chemicals, Loughborough, Leicester- s h e . Spectrophotometric grade methanol was purchased from Sigma Chemical Company Ltd. (Poole). Acetonitrile and 2-propanol (HPLC grade) were obtained from Rathburn Chemicals Ltd., Walkburn, Scotland. Buffer salts (i.e., sodium dihydrogen phosphate and disodium hydrogen phosphate) were supplied by Sigma. AG was a gift from Ciba- Geigy, Basle and was resolved by the method of Finch et al. l1

u-Glucose-6-monophosphate and NADP (monosodium salt) were obtained from Sigma and u-glucose-6-phospate dehydrogenase from Boehringer-Mannheim (Lewes, East Sussex). [ 1 p-”HIAndrostenedione (24 Ci/mmol) was purchased from New England Nuclear, Boston and [26, 27-3H]hydroxycholes- terol(83 Ci/mmol) from Amersham International (Little Chal- font, Bucks, U.K.). Scintillation fluid was Instagel from the Packard Instrument Co., Illinois.

Received for publication October 28, 1994; accepted February 2, 1995. Address repnnt requests to Dr. H. John Smith, Welsh School of Pharmacy, University of Wales College of Cardiff, Cardiff, CF1 3XF, Wales, United Kingdom.

Q 1995 Wiley-Liss, Inc.

ENANTIOSELECTIVITY OF AROMATASE INHIBITORS 377

All optical rotation measurements were performed with a Bellingham and Stanley polarimeter using 1% methanolic solutions. All melting points were determined using an electro- thermal melting point apparatus and are uncorrected.

P450AKOM and P45OcScc enzymes were prepared from human term placental tissue and bovine adrenal gland following the general method of Thompson and S i i t e~ i '~ and Hochberg et aI., l5 respectively.

The (?)-compounds (1)-(5) have been previously synthe- sised in our laboratories12 and the resolution of (1) and (3) into the (+)- and (-)-free bases via the (+)-and (-)-tartrate salts has been described. 'I3

(+)- and (-)-l-methylS-(4-aminophenyl)pyrroroli- dine-2,5-dione (2). A mixture of (+)-(2) (3.06 g, 0.015 mol) was stirred with (+)-tartaric acid (1.176 g, 0.0075 moll in methanol (80 ml). This mixture was heated until complete dissolution and allowed to cool on ice. The (+)-tartrate salt (2.1 g) was precipitated as a yellowlbrown crystalline solid. The (-)-tartrate salt was recovered by addition of (-)-tartaric acid to the mother liquors, and was again precipitated as a yellowlbrown solid (1.59 g). Both salts were separately recrystallised from methanol until steady-state optical rotation was achieved (seven recrystallisations). The free bases were recovered from aqueous solutions of the respective salts by the addition of an equimolar amount of aqueous sodium carbonate to yield 0.98 g of the (+)- and 0.95 g (-)-free base; (+)-form mp 165.4-165.6"C, [a]2su + 144.3", (-)-form mp 165.4-165.6"C, [a]251) - 142.5".

( + )- and (- )-3-(4-aminophenyl)-1,3-dimethylpyr- rolidine-2,5-dione (4). (+)-(4) (2.9 g, 0.013 mol) was dissolved in a methanolic solution of (-)-tartaric acid using the method previously described for (2). The (-)-tartrate was precipitated as a buff coloured crystalline solid (1.9 g). The (+)-tartrate was precipitated from the mother liquors yielding 1.84 g. Seven consecutive recrystallisations of the two salts, followed by reaction with sodium carbonate gave the (- 1- and (+)-free bases (0.69 g) and (0.74 g), respectively; (+)-form mp 123-125"C, + 79.6", (-)-form mp 123-125"C,

(+ )- and ( - )-3-(4-aminophenyl)-l,3-dipentylpyrroli- dine-2,5-dione (5). (+I451 (3.5 g, 0.010 moll was reacted with tartaric acid by the method previously outlined for (2), except that ethanol was used as the solvent. The (-)-tartrate (2.4 g) was precipitated on cooling as a brown solid. The (+)-tartrate (2.5 g) was recovered from the mother liquors on addition of (+)-tartaric acid. Six recrystallisations from ethanol yielded 0.98 and 0.88 g of the respective free bases after addition of sodium carbonate; (+)-form mp 78.4-79.2"C, [alZSLu +68.2", (-)-form mp 78.4-79.2"C, [a]25u -67.9".

[01]2") - 79.6".

Liquid Chromatography A Milton-Roy LC system was used consisting of a Model

3000 Constametric pump, a Rheodyne injection unit, and a Model 3100 variable wavelength spectromonitor. A Model CL-4100 computing integrator was used to process the data.

(*)-(2), (4), and (5) were separated on an a,-acid glyco- protein column (Chromtech AI3; 100 x 4.0 mm) and a precol- umn (10 x 4.0 mm) both packed with identical material with a mean particle size of 5 pm. A heated column jacket was

supplied by Jones Chromatography (Hengoed, Mid Glamor- gan) to standardise temperature. Injection on the column was achieved using a Hamilton syringe (50 pl) into a Rheodyne 20

Isocratic chromatographic conditions used were as follows: Mobile phase, (2), (4), and (5): 0.01 M phosphate buffer pH 7.4 containing for (2), 5% 2-propanob (4) 8% acetonitrile; (5) 13% acetonitrile. Flow, 0.5 ml min-'. UV detection, 250 nm. Temperature, 23°C. Injection volume, 20 p1. Pressure, 260 psi.

The (?)-forms of (2), (41, and (5) gave clean chromato- grams with clear separations of the (+I- and (-)-forms. The enantiomers of (21, (4), and (5) similarly gave clean chro- matograms with a single elution peak. The peaks due to the (+I- and (-)-forms in the (+)-chromatogram were identified by spiking the racemate with known concentrations of one of the enantiomers. Under the chromatographic conditions used the elution times for the (+I- and (-)-forms, respectively, were (2) 1.86 and 2.64 min; (4) 6.37 and 3.98; (5) 4.57 and 6.89 min.

Spectral Binding Studies Preparation of rat liver microsomes A male rate (350

g) was killed by cervical dislocation and the liver removed, blotted dry, and weighed (16.4 g). The tissue was then added to four times its weight of 0.25 M sucrose, i.e., a 20% w/v homogenate. The tissue was finely chopped with scissors and homogenized with a Potter-Elvehjem homogenizer such that no pieces of tissue were evident. The homogenate was then centrifuged in a 10 x 50 ml rotor at 14,000 rpm (12,500g) for 15 min. The supernatant was removed, the cellular debris discarded, and the supernatant recentrifuged at 35,000 rpm (100,OOOg) for 60 min. The microsomal pellet was removed, resuspended in phosphate buffer (30 ml), then recentrifuged as before. Finally the washed microsomes were removed and suspended in a minimum volume of buffer (10 ml), and aliquots (1 ml) of the suspension were transfered to plastic vials (1.5 ml) and stored at -20°C.

Ks determination16 Two aliquots (1 ml) of the microso- ma1 suspension were thawed and added to 0.1 M Tris buffer (pH 7.4, 10 ml) to give a protein concentration of 1.33 mg ml-' for rat liver microsomes and 3 mg ml-' for placental microsomes. Aliquots (3 ml) of the suspension were added to 3 ml matched quartz cuvettes and the spectrophotometer calibrated. Aliquots of test compounds dissolved in DMSO (10 pl) were added to the suspension in the sample cuvette, to give a final test compound concentration of 1-7 mM, and a similar volume of DMSO was added to the reference cuvette. The absorbance of the solutions relative to each other were then scanned from 500 to 360 nm on a Shimadzu 3000 spectrophotometer (V.A. Howe, Banbury, Oxon). Further 10-p1 aliquots of the compound were subsequently added and the procedure was repeated (Fig. 1).

A double reciprocal plot of the absorption change versus substrate concentration gave a linear curve intersecting the x-axis at - l/Ks (Fig. 2).

Enzyme Inhibition Studies P450AROM assay The method used was measurement of

'H20 released from [l P-'Hlandrostenedione by the general

p,I loop.

378 PEPPER ET AL.

Fig. 1. Spectral changes induced by (-)-(3). Placental protein was 3 mg ml- ' and incubation conducted with ligand concentration from 1-7 mM.

method of Graves and Salhanick. [ 1 p-'H]Androstenedione (75 pl, 100 pCi/ml) was transferred to a glass vial and the solvent was removed under a stream of nitrogen. A solution of unlabelled androstenedione (0.026 mM) in propane-1,2-diol(l ml) was added to the radiolabelled residue in the vial and mixed well to give a final concentration of 20 phi' for androstenedione. Incubations (0.5 ml) in phosphate buffer (50 mM, pH 7.4) containing microsomal protein (10 p1, 0.2 mg/ml final concentration) and NADPH-generating system (50 pl), consisting of NADP (1 mM), ~-glucose-6-phosphate (10 mM), and ~-glucose-6-phosphate dehydrogenase (4 SI/ml) in phosphate buffer (50 mM, pH 7.4) were warmed to 37°C in a shaking water bath for 5 min and the substrate solution (10 p1) was added to give a final substrate concentrations of 0.4 phi'. An aliquot (300 ~ 1 ) was removed and added to a cold mixture of 1% activated charcoal (900 pl) and mercuric chloride (1 mM, 300 pl). The mixtures were allowed to stand on ice for 20 min and were then centrifuged at 3000 rpm for 20 min. Aliquots (500 pI) of the supernatant were dispersed in Insta- gel (3 ml) and counted for 5 min on a LKB Wallac Rackbeta liquid scintillation counter (Crownhill, Milton Keynes).

IC5,, determinations The assays were camed out with a single substrate concentration of [lp-3Hlandrostenedione (0.4 phi") and an &bitor concentration range of 1, 5, 10, 25, 50, and 100

An IC,, value for each compound tested was obtained graphically from a plot of percentage of enzyme activity re- maining, against log,, inhibition concentration. Regression lines were calculated by least-squares analysis. Aminoglute- thunide was included in the test for comparative purposes.

P450cscc assay The method used was measurement of ['4C]methylpentanal released from [26, 27-'H]hydroxycholes- terol by the general method of Hochberg et al. l5

The bovine mitochondria1 suspension was thawed under cold running water, and sonicated, on ice, using a probe soni- cating disintegrator at 25 pm, for 3 x 10 sec, interspersed with 30 sec cooling intervals. The NADPH-generating system was prepared consisting of NADP (0.001 M ) , u-glucose-6- phosphate (0.01 M ) , and ~-glucose-6-phosphate dehydrogenase (4 Wml of generating system) in the incubation buffer (pH 7.4).

in the final incubation volume (0.5 ml).

TABLE 1. Spectral binding and enzyme inhibition parameters for the enantiomers and racemates of a series

of 1- and 1,3-substituted 3-(4-aminophenyl)-pyrrolidine-2,5-diones and AG

p450AROM p450cscc

Ks (CLM) % inhibitionb Compound IC," (rn (l)-(k) 17.20 (k0.5) 1.72 (20.3) 21 (21.2)

(+I 7.96 (k0.5) 0.96 (20.1) 19 (51.3) (-) <lo0 (20.5) 3.01 (t0.1) 24 (21.6)

(214 2 ) 16.42 (20.5) 4.61 (k0.2) 21 (21.4) (+I 7.31 (20.5) 2.46 (k0.4) 22 (21.3) (-1 <lo0 (k0.5) 5.85 (k0.Z) 16 (Ll.0)

0.81 (20.5) 1.96 (20.1) 29 (21.7) 12.1 (20.5) 0.37 (20.5)

16.72 (k0.5) 7.48 (20.5)

<lo0 (k0.5) 1.20 (k0.5)

15.6 (k0.5) 0.43 (20.5)

13.53 (k0.5) 6.01 (k0.5)

<lo0 ('0.5)

2.94 (20.3) 0.81 (20.1) 6.31 (k0.2) 5.12 (k0.1) 7.43 (k0.2)

16.3 (20.2) 18.4 (k0.2) 12.4 (k0.3) 1.26 (20.1) 0.85 (20.2) 3.47 (20.1)

31 (21.4) 35 (21.3) 22 (21.6) 17 (22.0) 20 (21.7) 12 (21.2) 15 (21.0) 12 (21.5) 70 (21.4) 73 (21.6) 66 (21.5)

"0.4 f l androstenedione. 'inhibitor = 100 pA4; t26.27 -.'H]hydroxyandrostenedione = 5 pA4.

The final incubation assay mixture (0.5 ml) consisted of incubation buffer (pH 7.4, 380 pl), [26, 27-3Hlhydroxycholes- terol as substrate (5 pM), NADPH generating system (50 pl), and inhibitor (100 pM) in DMSO where required. The tubes were warmed to 37°C for 5 min and the assay was started by addition of sonicated enzyme 0.2 mg/ml (10 1.1) which has been preincubated for 2 min at 37°C. The reaction was arrested after 15 min by pipetting a 400-p1 aliquot into NaOH (0.1 ml). The mixture was extracted with chloroform (1 ml), and aliquots of the aqueous layer (250 p1) were added to tubes containing 0.25 g alumina and 250 p.1 deionised water. These tubes were then centrifuged for 20 min at 3000 rpm (2000g). Aliquots (250 pl) were then dispersed into scintillation fluid (3 ml) and counted for 5 min each. The results are expressed as a percentage inhibition of the enzyme compared with a control value. Aminoglutethimide was included in the tests for comparative purposes.

RESULTS In the spectral bindmg studies interaction between the cy-

tochrome P450 of rat liver and human placental microsomes with the (+)-, (-)-, and (*)-forms of all the compounds tested, elicited Type 11, low-spin, difference spectra with peaks at about 420nm and troughs at about 390m (Fig. 1).

Quantitative studies with the human placental enzyme showed that although these interactions were qualitatively simdar, the strength of binding, expressed as Ks values (Fig. 2), were sigruficantly different (Table 1). For (l), (2), and (4) the (+I-enantiomer gave the lowest Ks value (i.e., strongest binding), whereas the (-)-enantiomers of (3) and (5) gave

ENANTIOSELECTIVITY OF AROMATASE INHIBITORS 379

3

::I, , / , , I , , , ~, , , , , , , , 0 -1.0-0.8-0.E-0.4-0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2

MSl (mM) Fig. 2. Double reciprocal plot determined for ( 2 )-AG-induced spectral changes

on incubation wrth human placental microsomes (3 mg ml I ) . The intercept on the x-axis gives the value of - l/K+ SD is within symbol unless otherwise shown.

lower K , values than either the (+)-enantiomer or the racemic mixture. In every case the racemic compound was not bound as strongly as the most effective enantiomer.

The IC,,, values for the inhibition of P45OAKOM indicate that stereoselection is occurring at the active site of the enzyme (Table 1). Compounds (l), (2), and (4) inhibit most effec- tively when the (+)-enantiomer is used, while for compounds (3) and (5) the (-)-enantiomer is the most potent.

Screening of compounds (1)45) as inhibitors of P450cscc showed that they were less potent than aminoglutethimide and the ( + I - , (-)-, and (?)-forms had about the same potency (Table l).

DISCUSSION The (+)-(R)-form of AG has been shown' to be 36 times

more potent than the (-)-enantiomer as an inhibitor of P450AKOM and this was confirmed by the present study. In- deed, stereoselective idubition was observed for all the compounds tested-about 30-fold difference between enantiomers and about 2- to 3-fold difference between the most potent enantiomer and the racemic mixture. However, whereas the (+)-enantiomers of (l), (2), and (4) were the active inhibitors, the (- )-enantiomen of (3) and (5) pos- sessed this role. It is concluded that 1-alkylation of the succin- h i d e ring with an alkyl group larger than 1-methyl caused an inversion of the stereoselective preference for the aromatase active site, i. e., the ( - benantiomer became more potent than the (+)-enantiomer.

We have suggested a model17 using molecular graphics for the binding of (+)-(R)-AG and a variety of other known inhibitors to the active site by superimposition on the substrate androstenedione in a manner such that N,, C, (C,) carbonyl of the inhibitor superimposed on the C, and C, carbonyl of the substrate, respectively. This places the phenylamine nitrogen above the nietabolised C10 methyl group of the substrate to ligate with the Fe:" haem of the cytochrome. From studies with substrate analogues the volume available to the 2-position is limited whereas there is space available at the 4-posi-

tion. The introduction of a small group on the glutarimide NH, e.g., CH, in AG, rogletimide, l8 and other related inhibitors, leads to a decrease in potency in accord with restricted access to the C, position of the substrate binding site as predicted.

of rogletimide, the pyridyl analogue of AG, is in accord with our model. However, place- ment of an octyl chain at N, of rogletimide leads to a switch over in enantjoselectivity to the (-)-(S)-form of the compound. Laughton et al. have accommodated this phenomenon within our model by suggesting alternative binding of the glutarimide ring to the active site. Rotation of the molecule through 180" on its 6-carbonyl group axis superimposes the N, at the C, position of the substrate and the bulky1 alkyl chain is accommodated by the space available at this position of the binding site. The (-)-6)-form ligands to the Fe"' haem in this case.

Modelling by Laughton et al.

0

I

Aminoglutethimide (1) : R - R' = H

(2) (3) : R = C5Hll, R' = H

(4) R = R' = CH3 (5) : R = R' = C5Hll

R = CH,, R'= H

(R)- (+) - ( I ) (S)-(-)-Octylrogletimide

The observation made here that the I-pentyl substituted compound (3) and the dipentyl compound (5) exhibit most of their inhibitory activity through the ( - )-enantiomers, whereas the 1-methyl, dimethyl, and the unsubstituted compounds show greatest potency with the (+)-enantiomers is readily accommodated within the modified model of Laughton et al. Since the 1-methyl substituted compound did not show this reversal in stereoselectivity (but had low potency) there

380 PEPPER E T AL.

would appear to be a critical chain length or steric bulk required to elicit this reversal in stereoselectivity.

The (+)-form of (2) provided crystals suitable for single crystal X-ray crystallography using an Enraf-Nonius CAD4 automatic diffractometer through the kindness of Professor M. B. Hursthouse at the SERC National Crystallographic Centre, UWCC and was found to have the (R)-configuration at C3.19 It seems that the (+)-forms of (1) and (3) will have a similar (R)-configuration based on the following argument. Hudson's rules" for the proximity effect on configuration of two asymmetric centres in sugars state that a rotatory contri- bution of one centre is only affected to a minor degree by changes in the structure of the remainder of the molecule provided the changes are not on contiguous atoms. Here the [01125D values for the (+)-forms of (l), (2), and (3) were +131.2", +144.3", and +161.2" in accord with the N, substit- uent (H, CH,, and C5Hll, respectively) having little effect since it is at some distance from the chiral centre. These conclusions account for the stereochemical activity profiles for compounds (l), (21, and (3) and add support to the views of Laughton et al. l8

In this present study, AG and the derivatives of 3-(4'- aminophenyl)pyrrolidine-2,5-diones were tested by spectral methods for their qualitative and quantitative reaction with cytochrome P450 in the form of rat liver microsomes and human placental microsomes. On interaction with P450, the (+I-, (-I-, and racemic forms of all the compounds tested elicited Type 11, low-spin, difference spectra with peaks at about 420 nm and troughs at about 390 nm. Type I1 binding spectra indicate an interaction of the primary amine of the compound with the cytochrome Fe3+ haem.21 Although these interactions were qualitatively similar, the strength of binding expressed as Ks values for the placental enzyme were signifi- cantly different. For (l), (2), (4), and AG the (+)-enantiomer gave the lowest Ks value (i.e., strongest binding), whereas the (-1-enantiomers of (3) and (5) gave lower Ks values than either the (+)-enantiomer or the racemic mixture. In every case the racemic compound was not bound as strongly as the most effective enantiomer. These results indicated the stereoselective nature of the P450AK()M active site for this group of compounds and qualitatively reflects the enantiomeric selectivity found from the enzyme inhibition studies. However, there was no correlation between K, and I&,, values within the series of compounds studied. This may be due to the use of a placental microsomal preparation containing a number of cytochrome P450s and the observed K , value reflects non-

(+)-AG is 2.5 times more potentz2 than the (-)-enantiomer as an inhibitor of P45OCsCC. In this work we found little enantioselectivity for the enzyme using AG and compounds (1145). However, the pyrrolidine-2,5-diones were less potent than AG and in view of the increased P45OAKOM: P45OcScc inhibitory ratio compared to that of AG they showed greater selectivity for P450AHOM.

Specific binding to P 4 5 0 ~ ~ 0 ~ .

ACKNOWLEDGMENTS We wish to thank the Cancer Research Campaign for sup-

porting the work of our group.

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enantioselectivity of aromatase inhibitors: substituted 3-(4-aminophenyl)pyrrolidine-2,5-diones

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