Benz[a]anthracene diols: Predicted carcinogenicity and structure-estrogen receptor binding affinity relationships

Benz[a]anthracene diols: Predicted carcinogenicity and structure-estrogen receptor binding affinity relationships Gregory M. Anstead* and Philip R. Kym'~ *Departments o f Internal Medicine and Pediatrics, Albert B. Chandler Medical Center, University of Kentucky, Lexington, Kentucky; "PDepartment of Chemistry, University of lllinois, Urbana, Illinois

Benz[a]anthracenes are ubiquitous environmental carcinogens that exert estrogenic and antiestrogenic effects directly or via hydroxylated metabolites. In this paper, the structure-estrogen receptor binding relationships of four 3,9-benz[a]anthracene diols are described: unsubstituted, 7-methyl, 12-methyl, and 7,12-dimethyl. Compounds unsubstituted at the 12-position have flat molecular topology, whereas methyl substitution at the 12position in the bay region induces twisting of the molecular framework. The oxygen-oxygen distances (11.9411.98 ,~) are similar to diethylstilbestrol (12.1 A). The binding affinities range from 0.43% to 26% that of estradiol. Methyl substitution at the 7-position enhances affinity; 12-methyl substitution decreases it. These results are contrary to many estrogen receptor (ER) ligand systems, in which the compounds with the flatter molecular geometries typically have lower binding affinity. Molecular graphics were used to analyze the fit of the four compounds with a receptor excluded volume model for the ER. These studies suggest that these compounds bind to the ER in a manner in which the anthracene fragment acts as the steroid AB-ring mimic (i.e, the benz[a]anthracene 9-position corresponds to the estradiol 3-position). Molecular orbital (AM1) calculations were used to calculate the charges of selected atoms. The 7-methyl compound was found to have greater charge similarity to estradiol than the other three compounds. The high affinity of the 7-methyl compound is ascribed to its charge similarity to estradiol, hydrophobic interactions in the receptor region that would accommodate a substituent in the planar 6-position of a A6"7-steroid, and favorable dispersive interactions with the receptor secondary to its extended planar system. Molecular orbital calculations also suggest that some of the benz[a]anthracene monophenols and diphenols have sufficiently low ionization potentials to act as carcinogens by a radical cation process. (Steroids 60:383-394, 1995) Keywords: orientation; carcinogenesis;ionization potential; environment;antiestrogen

Introduction There is concern that environmental estrogens may be contributing to the increasing incidence of breast cancer and disorders of the male reproductive tract. 1-4 Benz[a]anthracenes are ubiquitous in the environment; the methylated analogs of benz[a]anthracene (1) are potent inducers of carcinogenesis 5-s and act as partial estrogens in pituitary and hypothalamic tissues9; their 3,9-diols (2--5) have been proposed as metabolites. 1o The presence of the two terminal Address reprint requests to GregoryM. Anstead, Departmentsof Internal Medicine and Pediatrics, Albert B. Chandler Medical Center, University of Kentucky, Lexington, KY 40536. Received August 25, 1994; acceptedNovember 10, 1994. Steroids 60:383-394, 1995 © 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

hydroxyl groups, as in estradiol and many nonsteroidal estrogens, prompted an investigation of these compounds as estrogen receptor (ER) ligands and as antiestrogens.t° The structure-receptor binding affinity (RBA) relationships of the benz[a]anthracene diols are interesting in that a suitably positioned methyl group can enhance or decrease binding affinity by 60- and 1 l-fold, respectively, lO The goals of this work are to: (1) develop a structural rationale for this methyl substitution effect; (2) examine the orientation of these unsymmetric ligands within the receptor binding site; and (3) predict the carcinogenicity of these and related compounds using calculated ionization potentials. The threedimensional structure of the hormone-binding domain of the estrogen receptor has recently been proposed in a low res-

0039-128X/95/$10.00 SSDI 0039-128X(94)00070-S

Papers olution, computer-derived model, 1= but otherwise is unknown. Thus, molecular graphics, excluded volume analysis, ~2 molecular orbital calculations, and empirical thermodynamic approaches are used to interpret the binding data.

region 12-methyl group is locked into position by steric interference with the ring proton at position 1, whereas the 7-methyl group is a free rotor, z° Thus, the 12-methyl group may require a more precise fit in a ligand-receptor interaction.

Experimental

Oxygen-oxygen distances

Molecular modeling and calculations

The pharmacophore of the ER is two hydroxyl groups separated by an intervening hydrophobic spacer. 21 There have been efforts to relate estrogenic activity and receptor binding affinity to the distance between the oxygens in ER ligands. 22 Using the molecular graphics constructs of the benz[a]anthracene diols, the O - O distances in those molecules were compared to estradiol and high-affinity nonsteroidal ER ligands (Table 1). The O - O distances in the benz[a]anthracene diols are very similar to DES 24 and hydrogen-bonded estradiol. 23 The twist in the benz[a]anthracene skeleton induced by the 7,12-substituents has little effect on the O - O distance. Thus, it seems possible that either a Type I or Type II benz[a]anthracene diol may be able to interact with the same hydrogen-bonding sites of the ER as steroidal and non-steroidal ligands.

Molecular graphics and calculations were performed on a Silicon Graphics Indigo Elan Computer. The SYBYL Molecular Modeling Package (Version 6.0, Tripos Associates, St. Louis, MO, USA) was used for molecular superpositions and volume calculations, as an interface for AM I semi-empirical molecular orbital calculations and the Cambridge Structural Database. Crystal structure data for estradiol, benz[a]anthracene, 12-methylbenz[a]anthracene, and 7,12-dimethylbenz[a]anthracene were obtained from the Cambridge Database and read into the MacroModel molecular mechanics program (Version 3.5). ~3 Appropriate group additions and deletions were made to the crystal structures by the MacroModel system to obtain structures 2--5; each structure was minimized by molecular mechanics to a gradient of <0.05 kJ/~ 2. The minimized structures were then taken into SYB YL version 6.0 where AM1 semi-empirical molecular orbital calculation¢ 4 were performed with full geometry optimization. The Precise option of the SYBYL Program was used to tighten convergence tolerances.

Results and Discussion

Molecular structures of benz[a]anthracenes Structurally, the benz[a]anthracene (BAs) fall into two classes: Type I, which are unsubstituted in the bay region (positions 1 and 12), and Type II, which are substituted at position(s) 1 and/or 12, and are distorted from planarity by steric interactions in the bay region. 15 Typical Type I and II BAs are compared in Figure 1. The compounds that have been examined as ER ligands fall into both classes. Each of these structural types will be compared to estradiol by molecular graphics techniques to determine which resemble the steroid more closely. For the Type I benz[a]anthracene diols (2 and 4), the X-ray crystallographic coordinates of benz[a]anthracene (1) were used, 16 with standard A r - O and A r - C H 3 bonds attached as needed. For the type II compounds (3 and 5), the crystallographic coordinates of 7,12-dimethylbenz[a]anthracene 17 and 12-methylbenz[a]anthracene 18 were used, with appropriate group attachments and deletions (Figure 2). In 7,12-dimethylbenz[a]anthracene, the angular ring is tilted 21 ° and the 12-methyl group 12° out of the plane of the anthracene system. 19 The 7-methyl group does not exert a large effect on the interring angles. In addition, the bay

Estrogen receptor binding affinity and structure-binding affinity relationships Morreal and coworkers reported the association constants of compounds 2-5 for the estrogen receptor from rat uterus (Table 2). 1o For simplicity, we convert these data to a ratio of association constants (RAC) (i.e., [Kassoc,cmpd/Kassoc.E2 ] 100%). Methylation at the 7-position leads to a 60-fold enhancement in RAC for 4 versus 2, but only a 3.9-fold enhancement for 5 versus 3. In contrast, methylation at the 12-position leads to an I 1-fold decrease in the RAC (5 versus 4) or a slight enhancement (3 versus 2). The effects of methylation on the RAC are summarized in Figure 3. Note that the effects of methyl substitution are not parallel (i.e., substitution at positions 7 and 12 produce different effects on the RAC, depending on whether or not substitution exists elsewhere). If the contribution of a substituent to the binding is not independent of the other groups present, this suggests that the substituent is exerting more than a local effect; it may be altering the orientation within the ER binding site or the electronic structure. 26

Comparison with other ER ligand systems The high estrogen receptor binding affinity of 7-Me-BA 3 seems anomalous for an extended, planar system. Figure 4 shows the typical pattern of binding affinity dimunition seen when comparing other planar ER ligands to their nonplanar

Figure 1 Relaxed stereoview of the overlay of Type I and Type II Benz[a]anthracenes (unsubstituted and substituted in the bay region, respectively). A five-atom fit of the D' and C' rings was used.

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Benz[a]anthracene dio/s as estrogen receptor/igands: Anstead and Kym 2 1

I1 0

12 ~ 4

9 " ~ 8

~

" 7

~

6

5

HO R2

1 (benz[a]anthracene)

HO~

O

2:Rz = R 2 = H 3: Rl = C H 3,R 2 = H 4: RI = H , R 2=CH 3 5: R] = R2 = CH3

H H

O

6 (DES)

~

OH

7 (indanestrol)

OH

R2 R

15, R = phenyl (RBA = 34% (mouse)) 16, R = I (RBA = 49% (rabbit)) Figure 2

17, 18, 19, 20,

R1= R 1= R1= R 1=

CH3

R2 = H Me, R 2 = H H, R 2 = M e R2 = Me

21,X =R 1 -- H; Y =OH 22, Y =R 1 =H,X =OH 23, X = H, Rz = Me, Y = OH 24, Y =H, R z =Me, Y = OH

Structures of benz[a]anthracene and its analogs and selected steroidal and nonsteroidal estrogens.

counterparts. 27-32 The 12-substituted BA systems 4 and $ are not planar because of the bay region methyl group, yet have a poor RBA. Thus, the benz[a]anthracene diols display an opposite pattern of structure-RBA relationships compared to many other ER-ligand systems.

groups in the molecule, with consideration of the loss of molecular degrees of freedom and translational/rotational entropy. The observed free energy of binding is given by equation 1:

Thermodynamic evaluation of receptor fit

The dissociation constant K d is obtained from Kassoc. The AVERAGE values are calculated by equation 2:

The complementarity of fit between a ligand and its receptor can be determined by an empirical thermodynamic approach. 33'34 In this procedure, the observed free energy of binding of the compound (AGob~d), calculated from the RAC, is compared to the sum of empirically-derived binding energies (AVERAGE, AGav) for all the functional

AGobsd = RTInK d

AGav = - 1 4 - 0.7nDo F + 0.7ncsp2 + 0.8ncsp3 + 2.5nOH where - 14 is the standard value for translational/rotational entropy loss and n is the number of specific functional Table 2

Table 1

Comparison of oxygen-oxygen distances in estrogen receptor ligands

Compound Benz[a]anthracene-3,9-diol (2) 7,12-Dimethyl benz[a]anthracene-3,9-diol (5) Estradiol hemihydrate (03-017 distance) Estradiol hemihydrate (03-OH 2 distance) Diethylstilbestrol (6) Indenestrol A (7)

Oxygen-oxygen distance (A) 11.94a 11.99a

10.93b'2a 12.1b'22 12.1b,24 11.6b'2s

"Determined from molecular graphics models, bMeasured from crystallographic coordinates.

Association constants, binding energies, and AVERAGE values

Compound 2 3 4 5

E2

K,,,oc a

RACb

Binding energyc

AGavg d

Difference e

3.0 × 107 4.3 x 107 1.8 x 109 1.7 x 10a 0.7 x 10TM

0.43 0.61 26 2.4 100

9.34 9.54 11.6 10.3 12.3

2.2 3.0 3.0 3.8 3.4

7.1 6,5 8,6

6.5 8,9

"Association constant for the estrogen receptor (reference 10). bRatio of association constants (Kassoccmpd/KanocE) X 100%. CBinding energy = AG = - R T In K=,=ocl °Calculatecl'from eq. 2 (reference 33). eBinding energy - AGavQ.

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385

Papers OH

HO 3, RAC = 0.61

N•=3.9

OH

,OH

CH3

HO 2, RAC = 0.43

60~ F2=

~

H / O

HO~

CH3 5, RAC=2.4 F4 = 0.092

CH3 4, RAC = 26 Figure 3 Changes in RAC produced by methyl substitution. The enhancement factor, F, is given by the ratio RAC methyl substituted cmpd/RAC protio cmpd. Since F1 ¢ F4 and F2 ¢ Fa, different orientations in the ER binding site are suggested.

groups or degrees of freedom (DOF). The values of AGousa and AGav are summarized in Table 2. The greater difference value of the 7-methyl substituted compound 4 compared to the unsubstituted compound 2 indicates that this substitution pattern produces an increase in binding affinity greater than that expected from methyl substitution alone. This provides additional evidence that 7-methyl substitution may be producing more than a local hydrophobic effect on the binding affinity; it may alter the

(

(

.of 8, RAC = 111 (L)

orientational and electronic features of the benz[a]anthracene diol system. Under optimal conditions, a methylene group (a methyl group minus the original hydrogen) may locally contribute 3.1 kcal/mol to the binding free energy of a ligand-protein interaction. 35 The contribution of the 7-methyl group in 4 (2.3 kcal/mol, AGobsd4 -- AGobsd2) is below this optimal value, but well above the typical value of 0.8 kcal/mol for the contribution of a methyl group to the binding free energy. Thus, we can not rule out that the

)

9, RAC = 11.2 (L)

HO~

11, RAC = 8.2 (R)

12, RAC = 0.049 (R)

10, RAC = 1.15 (L)

O

H

13, RAC = 128 (L)

HO~

O

H

14, RAC = 7.6 (L)

Figure 4 Effect of planarity on ER binding affinity. RAC = ratio of association constants; L = lamb uterine cytosol; R = rat uterine cytosol. Compounds 8-10 (ref. 27), 11 and 12 (ref. 28), and 1:3 and 14 (ref. 29).

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Benz[a]anthracene diols as estrogen receptor ligands: Anstead and Kyrn 7-methyl substitution effect in enhancing the binding affinity of 4 is only a local hydrophobic effect, but if it is, it is atypical. The difference value of the 7-methyl compound 4 is only slightly less than that of estradiol, indicating a comparable degree of complementarity of fit within the ER binding site. By contrast, unsubstituted benz[a]anthracene diol 2 and the 12-methylated compounds 3 and $ have much lower difference values and poorer receptor fits. "The free energy of host-guest binding is the fundamental thermodynamic index of molecular recognition", 36 and has four components: (1) hydrophobic contribution; (2) dispersive interaction; (3) electrostatic interaction; and (4) steric repulsion. 37 The hydrophobic effect is proportional to the apolar surface area (APSA) of the ligands, 3s which has been calculated for estradiol and benz[a]anthracenes 2-5 using the sum of the Bondi surface areas 39 of the hydrocarbon groups of the molecules (Table 3). The APSA of the highest affinity benz[a]anthracene 4 is 75% that of estradiol, however, the binding free energy is 94% of that of estradiol. This suggests good complementarity of 4 with the ER in terms of dispersion, electrostatics, and sterics. Conversely, for the poor affinity analog 3, the APSA is 82% of estradiol, but the binding free energy is 78% that of estradiol, indicating lesser complementarity with the receptor. The maximum contribution of the hydrophobic effect to the binding free energy (AGhydroph)can be calculated, using the relationship of a gain of 46 cal//~ 2 of APSA. 4° For estradiol, the AGhydroph is calculated to be 12.0 kcal/mol, compared to the AGobsdof 12.3 kcal/mol. For the 7-Me BA 4, AGhydroph is 9.03 kcal/mol, versus AGobsdof 11.6 kcal/ mol. This indicates that in 4 there must be significant nonhydrophobic contributions to the binding free energy.

binding site of unknown topology, using known binding or bioactivity data and the three-dimensional structures of a known set of ligands. In E 2, the 3-OH group is more important for ER binding than the 1713-OHmoiety, 46'47 and thus the former is used as a common anchor point for the molecular graphics alignment of E2 with a phenol of the benz[a]anthracene diols. 25'41-4T There are four orientations that a benz[a] anthracene diol may adopt with respect to estradiol in the ER binding site (Figure 5). However, orientations II and IV are dismissed immediately because significant molecular bulk is presented near the region of the receptor that would be occupied by the C- 1 substituent of a steroid, a position of known steric intolerance. That is, the 1-hydroxy, 41'48 1-bromo, 49 1-methyl, 5° and 1,11-ethanoestradio151 all have poor binding affinity. To evaluate the fit of the BA ligands in the ER binding site, three criteria were used: (1) intersection of the molecular volume of the ligand with the RExV; (2) lack of interference with the receptor essential volume (REsV), that region of space occupied by the receptor itself; and (3) correspondence of the hydroxyl groups of the ligand with those of estradiol. To quantify the first criterion, the intersection of molecular volume of each compound in each orientation with the RExV was calculated. The RExV of the ER was created by the union of the molecular volumes of estradiol and five high-affinity estradiol analogs (lll3-ethyl-E2, 17ot-ethynyl-E2, 16~-bromoOH

Orientation There is considerable interest in the orientation that unsymmetric nonsteroidal ligands can assume in the ER binding site. 25'41-43 The orientation preferences of the BAs with the ER were analyzed with the receptor mapping technique (Active Analog Approach, excluded volume analysis). ]2'44'45 In this method, the correspondence of the hydroxyl groups of the BA and E 2 and the molecular volume that the BA shares with a composite volume of high-affinity estradiol analogs (the receptor excluded volume, RExV) are evaluated. The Active Analog Approach is useful to derive information about the fit of a ligand in a macromolecular

Estradiol (Ez) OH

HO

HO' D'C'/AB mode (Orientation D

(D'C/AB) 180"mode (Orientation ID

Table 3 Apolar Bondi surface areas a and molecular volumes~ of estradiol and benz[a]anthracene diols

Estradiol BA-3,9-diol (2) 12-Me-BA-3,9-diol (3) 7-Me-BA-3,9-diol (4) 7,12-diMe-BA-3,9-diol (5)

Apolar surface area (A 2)

Molecular

volume (A3)

261.3 179.0 196.3 196.3 213.6

244.5 208.9 220.9 223.5 232.4

aSum of the Bondi surface areas for the carbon-hydrogen groups in the molecule, bDetermined using molecular graphics.

HO~

O

H HO~

A'B'/AB mode (Orientation UI)

O

H

(A'B'/AB) 180"mode (Orientation IV)

Figure 5 Orientations that benz[a]anthracene-3,9-diols may adopt in the binding site of the estrogen receptor relative to estradiol.

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Papers Table 4 Molecular overlays of benz[a]anthracene ER ligands in specific orientations with the ER excluded volume; intersection of molecular volume, excess volume, and distance between oxygen Orientations (roman numerals)

Ligand Compound/Isomer BA-3,9-diol (2) 12-Me-BA-3,9-diol (3a) 12-Me-BA-3,9-diol (3b) 7-Me-BA-3,9-diol (4) 7,12-diMe-BA-3,9-diol (5e) 7,12-diMe-BA-3,9-diol (5b)

Intersection and (excess) volume (A 3)

I 182.0 191.4 182.2 184.6 194.6 185.1

(25.5) (27.6) (34.8) (38.0) (39.9) (45.7)

E z, 7cx-pentenyl-E2, and 15,16or-methylene-E2). 52 This union of all high-affinity analogs and the parent ligand represents that region of the binding site sterically accessible, and thus not occupied by the receptor itself. Another consideration is that compounds 3 and 5, substituted in the bay region, actually exist as two isomers, because of the helical twist of the carbocyclic array. 53 Each of these isomers is considered in the receptor mapping analysis. A large volume overlap (intersection) between the ligands 3-5 and the RExV implies a good fit in the ER binding site. By contrast, the REsV is the union of inactive analogs minus the RExV. This affords a region of space occupied by the receptor itself; these compounds are inactive because they sterically interfere with the REsV.

Overlays of benz[a]anthracenes with the receptor excluded volume The superficial resemblance of benz[a]anthracenes to steroids has long been recognized. 54 The BAs 2-5 were overlapped with the RExV in orientations I and Ill, and an intersection volume and the distances of the BA OH group from the 1713-OH of E 2 were calculated. In addition, the excess volume of the ligand (that outside the RExV) was determined (Table 4). The intersection volume may be considered as proportional to a polarizability volume available for dispersive interactions. 5 . ~ ,The excess volume represents possible areas of steric interference with the REsV. 55 The REsV map has not yet been formally created, and at this time we must rely on comparisons to substituted estradiols with low RBA to determine areas of probable steric repulsion with the receptor. In general, orientation I produces greater intersection volume, except for isomer a of 12-Me-BA 3, and closer distance to the 1713-OH, except in the b isomer of 12substituted compounds 3 and 5. In orientation I, the Type 1 BA diols 2 and 4 position their excess skeletal bulk primarily outside the BCD bay area of the ER RExV (Figures 6A and 6C). Part of this volume would be sterically unfavorable, based on the low affinities of the 15~x- and 15-13methylated estradiols (26% and 29%, respectively). 5o However, much of this region of space has not been explored for steric tolerance by substituted estradiols. In orientation III, the Type I BA diols have their excess skeletal volume outside the CD core of the RExV (Figures 6B and 6D), adjacent to positions 12, 13, and 18 of the steroidal RExV. On

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III 172.7 (34.5) 193.0 (35.1) 177.4 (50.9) 181.5 (39.3) 184.5 (52.0) 165.2 (42.8)

Distance from 1713-0H (A)

I 2.31 2.55 4.15 2.31 2.55 4.15

III 2.87 3.11 2.04 2.87 3.20 2.04

estradiol, 12-methyl substitution is well tolerated (RBA = l 1 !%), 56 but 18-methyl substitution is not (RBA = 31%) .56 For the 7-methyl compound 4 in orientation I (Figure 6C, the methyl group is in a position analogous to a planar 6-substituent on a steroid. Relatively large substituents in the planar 6-position of estradiol are well tolerated, as in 6-iodo-A6"7-estradiol (15; RBA = 49%) 57 and 6-phenylA6'7-estradiol (16; RBA = 34%). 58 (The Taft steric parameters E s are - 1.24 for a methyl group, - 1.40 for an iodine, and - 3 . 8 2 , - 1.01 for a phenyl group.) 59 For the 12-methyl compound 3, in Fit I, the methyl group occupies a position similar to the 11-carbon of the steroid (Figures 7A and 7C). For 3a, this fit provides a higher intersection volume and lower excess volume than the corresponding fit of the 7-methyl analog, and the distance from the 1713-OH position to the proximal hydroxyl groups of the two compounds is similar as well. NevertheA. (2-I)

B. (2-Ili)

C. (4-I)

D. (4-1II)

/-

Figure 6. Type I benz[a]anthracene diols 2 and 4 in orientations I and III relative to the estrogen receptor excluded volume. The grid represents molecular volume in excess of RExV.

Benz[a]anthracene diols as estrogen receptor ligands: Anstead and Kym B. (3a-III)

A. (3a-I)

/

C. (3b-I)

B. (5a-III)

A. (5a-I)

/

D. (3b-III)

D. (5b-III)

C. (5b-I)

"U Figure 7. 12-Methyl benz[a]anthracene 3 isomers a and b, in orientations I and III relative to the estrogen receptor excluded volume.

less, the binding affinity of the 12-methyl compound is lower. Fit I for the b enantiomer of 3 (Figure 7C) produces overlap comparable to the 7-methyl compound, but much poorer proximity of its second OH group to the 1713-OH site. Also, much of the excess volume of 3b lies in areas of known steric intolerance above the D-ring (1513, 1613, 18). 50,56 In Fit III, 3a also has a large intersection volume, but poorer 1713-OH correspondence as compared to Fit I (Figure 7B). The excess volume of the 12-methyl group lies near the 1113-position and is probably well tolerated. Additional excess volume is disposed similarly to BA-diol 2 in Fit III. However, the 3b isomer in Fit III has low intersection volume and high excess volume (Figure 7D). The 7,12dimethyl compound 5a in Fit I has the highest intersection volume and a close fit of its proximal hydroxyl to the 1713OH site (Figure 8A). Its excess volume is spatially disposed in a manner similar to a composite of 3a and 4 in Fit I. Fit III for 5a is not as optimal in terms of all the overlap parameters (Figure 8B). As for 3b, 5b in either Fit I or III has either poor intersection volume or poor hydroxyl correspondence (Figures 8C and 8D). Previously, on the basis of simple overlap of the crystal structures of BA diol and estradiol, Glusker had proposed that BA diols orient as in Fit III. 54 Our conclusion from the volume mapping analysis is that BA-diol 2 and its 7-methyl congener 4 probably orient as in Fit I, based on high intersection volume, low excess volume, close hydroxyl correspondence, and absence of severe steric restrictions. Both compounds with a 12-methyl group have an isomer (b) with either low intersection volume (Fit III) or poor hydroxy alignment (Fit I). Nevertheless, the a isomers of 3 and 5 do have high intersection volume and good hydroxy group cor-

Figure 8. 7,12-Dimethyl benz[a]anthracene diol 5, isomers a and b, in orientations I and Ill relative to the estrogen receptor excluded volume.

respondence in Fit I, so what explains the poorer RBA of the 12-methyl compounds? Even if the b isomers had negligible binding it would not totally account for the lower affinity of these compounds. Thus, we examined the electronic properties of these molecules as well.

Electronic structure of estradiol and benz[a]anthracenes The electronic compatibility of an ER ligand with its receptor depends on the presence of a phenolic ring and, to a lesser extent, on a second hydroxyl group. The phenolic OH group acts as a hydrogen bond donor or acceptor, but the aromatic ring itself is also essential to the RBA. 56 The carbons of the arene may function as a "hydrophobic negative charge" in the molecular recognition process 60 and may associate with positively-charged receptor sites or with aromatic amino acid residues of the ER in edge-to-face stacking interactions. The 6 + hydrogens on the aromatic ring may engage in "nontraditional" hydrogen bonds with receptor heteroatoms; these weak interactions m6aYcontribute 1-2 kcal/mol to the free energy of binding. The BAs, with less potential for hydrophobic interactions than estradiol due to lesser numbers of apolar hydrogens, may have greater capacity to engage in such weak interactions because of their polycyclic aromatic structures. Furthermore, unlike simple arenes (as in E2) which tend to edge-to-face stack, the extended planar array of the BA system makes face-to-face stacking more favorable. 61 To compare the electronic structures of the BA systems and E 2, the atomic charges were calculated by the AM1 semiempirical molecular orbital method. 14 The results appear in Table 5. The molecular graphics overlay procedure affords a spatial fit of the electronic features and polarizable mo-

Steroids, 1995, vol. 60, May

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Papers Table 5

Net atomic charges on selected atoms of estradiol and benzanthracenes 2-5 a

H 8

.o- "T" T"

7

H

Atom

E2

BA-diol (2)

12-Me-BA-diol (3)

7-Me-BA-diol (4)

7,12-diMeBA-diol (5)

C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 H1 H2 H3 03 03H O×b O×H

-0.0912 -0.2151 0.0782 0.1617 - 0.0293 -0.1237 -0.1522 -0.0880 -0.0555 -0.0925 0.1328 0.1321 0.1487 -0.2527 0.2168 -0.3201 0.1992

-0.0815 -0.1874 0.0739 -0.1605 - 0.0031 -0.1126 0.0296 -0.0163 -0.1019 -0.0590 0.1360 0.1333 0.1513 -0.2496 0.2180 -0.2468 0.2196

-0.0919 -0.1393 0.0864 -0.2210 0.0037 -0.1226 - 0.0264 -0.0178 0.0352 -0.0582 0.1377 0.1509 0.1351 -0.2498 0.2192 -0.2498 0.2138

- 0.0831 -0.1866 0.0708 -0.1606 0.0031 -0.0446 -0.0325 -0.0120 -0.1088 -0.0567 0.1348 0.1351 0.1519 -0.2524 0.2176 -0.2501 0.2187

-0.0923 -0.1393 0.0843 -0.2215 0.0032 -0.0562 -0.0214 -0.0155 -0.0422 -0.0558 0.1375 0.1519 0.1355 -0.2501 0.2185 - 0.2508 0.2181

aFrom AM1 calculations, b017 of E2, 03 of the benzanthracenes.

lecular volumes of the benz[a]anthracenes with E 2, whereas the atomic charges provides an indicator of possible electrostatic similarities. Table 5 summarizes the charges on the atoms of the AB-rings of E 2 and D'C'-rings of the benz[a]anthracenes. These are the ring systems proposed to coincide based on the molecular overlap studies (i.e., BAdiols in orientation I relative to E2). In comparing the flat BA-diols 2 and 4 with the twisted analogs 3 and 5, it is notable that the charge patterns of C-2 and C-4 are inverted. Thus, compounds 2 and 4 have greater charge similarity at the C-2 and C-4 positions to E 2. Furthermore, 7-Me-BAdiol 4, the BA compound with the highest RAC, also has greater charge similarity to estradiol at positions H 1, H2, H4, 03, and O3H than the twisted BA analogs. Overall, the charge patterns of the phenol and A-ring pharmacophores of E z and 7-Me-BA-diol are quite similar. The calculations also reveal that the oxygen of the 1713-OH group of E2 bears a greater negative charge than the corresponding oxygen (09) of the BA-diols. The 1713-OH hydrogen has a lesser positive charge than the O9H hydrogen. These charge difTable 6

ferences may affect the ability of the BA-phenol (and phenols in general) to act as a 1713-OH mimic.

Ionization potentials Ionization potential (IP) and its negative, the energy of the highest occupied molecular orbital, is a measure of the electron-donating capability and hydrogen-bond basicity of a molecule, and has been used as a parameter in quantitative structure-activity relationship studies. 62'63 The BA diols have considerably lower ionization potentials than estradiol (see Table 6). Like the simple BAs, in which methyl substitution decreases the IP,~gthe AM1 calculations suggest that in the BA diols, methyl substitution also decreases the ionization potential. The AM1 method is reported to reliably reproduce the trends in ionization potential for conjugated molecules, but overestimates it. 64 The 7-Me-BA-diol 4 and the 7,12-di-Me-BA-diol 3 have nearly identical calculated ionization potentials (and EHOMO) but differ in RAC by 1l-fold, suggesting IP is not an important determinant of RBA.

Ionization potentials (eV) of benz[a]anthracenes and hydroxybenz[a]anthracenes

Estradiol BA-3,9-diol (2) 12-Me-BA-3,9-diol (3) 7-Me-BA-3,9-diol (4) 7,12-diMe-BA-3,9-diol (5) Benz[a]anthracene (17) 12-Methylbenz[a]anthracene (18) 7-Methylbenz[a]anthracene (19) 7,12-Dimethylbenz[a]anthracene (20) 7-Methylbenz[a]anthracene-3-ol (21) 7-Methylbenz[a]anthracene-9-ol(22) 7,12-Dimethylbenz[a]anthracene-3-ol (23) 7,12-Dimethylbenz[a]anthracene-9-ol (24)

IPcTa

IPPES b

IPAM1c

IPAM1,corrd

NA e NA NA NA NA 7.54 7.38 7.37 7.22 NA NA NA NA

NA NA NA NA NA 7.46 7.27 7.27 7.10 NA NA NA NA

8.86 8.13 8.11 8.04 8.01 8.205 8.097 8.094 7.991 8.094 8.055 8.008 8.025

8.52 7.43 7.40 7.29 7.25 NC f NC NC NC 7.37 7.32 7.24 7.27

aFrom the absorption m a x i m u m of the charge-transfer complex with chloranil, bFrorn photoelectron spectroscopy. CFrom AM1 calculations. °The corrected AM1 ionization potential, using eq. 9. eNA = not available, fNot corrected, because experimental values are available.

390

S t e r o i d s , 1 9 9 5 , vol. 60, M a y

Benz[a]anthracene diols as estrogen receptor ligands: Anstead and Kym To support the reliability of the calculated ionization potentials of the BA diols, the AM1 method was used to calculate the ionization potentials of similar compounds, the simple BAs 17-21. These calculated IP values may then be compared to available experimental values. 19.65 The AM1 calculations on the simple BAs 17-20 reproduce the trends in IP well, but, as expected, the values are too high. In the manner of Anderson et al.,66 the calculated ionization potentials were related to the experimental values obtained from photoelectron spectroscopy 19 and charge-transfer complexes, 65 using simple regression equations: IPAM1 = 3.78 + 0.594 IPpzs (r2 = 0.999)

(9)

IPAM1 = 3.17 + 0.668 IPcT (rz = 1.00)

(10)

and

Using equation 9, it is now possible to correct our AM1 calculated ionization potentials for E z, 2, 3, 4, and 5 to 8.52, 7.43, 7.40, 7.29, and 7.25 eV, respectively.

Ionization potentials and carcinogenesis The corrected ionization potentials allow a prediction of these compounds as carcinogens via a radical cationic process. Cavalieri and Rogan report that only polycyclic aromatic hydrocarbons with an IP below 7.35 eV can be metabolically activated to carcinogens by one-electron oxidation. 65 Thus, one could predict that 4 and 5 may act as carcinogens in this manner, whereas 2 and 3 cannot. It is of interest to extend this analysis to the monophenols of the benz[a]anthracenes (21-24), because these compounds are known metabolites. All of these compounds have corrected ionization potentials of 7.37 eV or less (see Table 6), suggesting that these may also be capable of acting as carcinogens via the one-electron pathway.

Polarizability model With the lower apolar surface area of the benz[a]anthracene diols compared to estradiol, a relatively high binding affinity compound such as 4 may require a significant contribution from dispersion forces. This dispersive interaction can be approximated as: O~R ~L

D ~

r6

(11)

where D is the dispersion energy, a R and 0% are the polarizability of the receptor and ligand, respectively, and r is the separation distance. 67 The twist of the BA skeleton in the 12-substituted compounds 3 and 5 forces the distance between the ligand and receptor apart and disrupts the mutual dispersive forces. Because the vdW force is proportional to 1/r°, even a small increase in ligand-receptor separation distance can appreciably affect the attainable dispersion interaction between ligand and receptor. This phenomenon was originally described for ortho-substituted polyhalogenated biphenyls binding to the aromatic hydrocarbon (Ah) receptor. Thus, for the benz[a]anthracene diols, binding affinity may also be dependent on the presence of an accessible planar face which can engage in dispersion interaction with the receptor. Figure 9 shows other essentially

OH

HO 25 (20%, calf)

26 (20%, calf)

Figure 9 Other dimensionally flat ligands with relatively high ER binding affinities.

planar compounds with significant estrogen receptor binding affinity.68'69 A similar polarizability effect may be operative for these compounds as well.

Unifying scheme for biological activity of benz[a]anthracenes and their phenolic derivatives The BAs and their metabolites have diverse biological effects including carcinogenesis, chromosomal alteration, changes in intercellular gap junctions and the cytoskeleton, and photochemical DNA scission. 7° Figure 10 provides a unifying hypothesis for the biological actions of BAs and their phenolic metabolites. In the well-established pathway of this scheme (top left), the BAs undergo initial oxidation to arene oxides, followed by hydration to dihydrodiols and further epoxidation to the well-known ultimate carcinogen, the diol epoxide. BA phenols arise from rearrangement of the arene oxides. 71 Interestingly, these BA phenols feedback-inhibit the initial epoxidation. 72 Alternatively, the BAs may act as carcinogens by a oneelectron oxidation to radical cations, catalyzed by peroxidases, including prostaglandin H synthase. 65 The calculations herein suggest that certain BA phenols and diphenols have sufficiently low ionization potential to also act as carcinogens in a radical cation process. The BA phenols are known metabolites; the diphenols 2-5 have yet to be isolated, but have been proposed as metabolites, lO Dimethylbenz[a]anthracene (17) is known to have weak affinity for the ER (0.001% of E2) and acts as a partial estrogen agonist. 9 Table 1 shows that BA diphenols 2-5 possess significant ER binding affinity. BA diphenols 2 and 5 have been found to have weak estrogenic potency (1/4464 and 1/4000 of E 2, respectively). 73'v4 Furthermore, BA diphenols 2-5 are weak antiestrogens; compound 5, the most potent, is 10 times weaker than nafoxidine. 2° Nevertheless, based on the molecular overlays of Figures 5, 6, and 7 the antiestrogenicity is surprising, since these compounds do not occupy the traditional "areas of antiestrogenic influence" off the 1113- and 7or-positions. 75'76 The ER binding affinity and estrogenicity/antiestrogenicity of the BA monophenols has not been investigated. On the other hand, BA diphenols 2-5 may act as antiestrogens indirectly, by binding to the Ah receptor, an intracellular protein widely distributed in mammalian cells with high affinity for polynuclear aromatic hydrocarbons (including dimethylbenz[a]anthracene). Ah receptor activation is thought to inhibit estrogen-stimulated gene transcription or induce estrogen metabolism. 77'78 Finally, the BA diphenols may be oxidized by prosta-

Steroids, 1995, vol. 60, May

391

Papers direetpaaial

~

cytochr

BAs

P-450

/

to ~ AhR

depletionof intrat~ular estrogens

al~n¢

/

dihydrodiols

[0! ?

\

~ diol - epoxides

~ carcinogenesis

peroxi,~s

le-

BA-ol (phenolic)

/

vpoxide

- oxides hydrase~

IP

radical c~ons

(peroxadases)

BA-diol

binds

J

1

antiestrogen effect

? pmstaglandinH

synthase

vicinalphenols

ortho-qtfinones

1

bind to maeromolecules Figure 10 Unifying scheme for the biological activity of benz[a]anthracenes and their phenolic metabolites.

glandin H synthase (PHS) to vicinal phenols, then to o-quinones, which can covalently bond to biological macromolecules. This route is proposed because numerous ER ligands are oxidized by PHS and this reaction is proposed to mediate the genotoxic and carcinogenic effects of estrogens. 79 Thus, it seems reasonable that the BA diphenols could also act as substrates for this enzyme. The significance of environmental estrogens in carcinogenesis is an area of controversy, s° The complexity is created by the high human background exposure to synthetic, environmental, and endogenous estrogens s° and the fact that individual compounds can exert multiple biological effects. For example, the antiestrogen tamoxifen has been advocated for the prevention of breast cancer, 81 but paradoxically may induce liver cancer. 82 The relationship between estrogen receptor binding and carcinogenesis remains uncertain. Xenoestrogens such as the benz[a]anthracene diols may affect the production and metabolism of other estrogens, such as 2-hydroxyestrone and 160t-hydroxyestrone.3 The former compound is weakly estrogenic and nongenotoxic, whereas the latter compound is a potent, genotoxic estrogen. Alternatively, Duax and coworkers s3 have proposed that the receptor transports the carcinogenic ligand to specific sites on DNA involved in steroid-regulated growth. If the receptor-bound carcinogen has exposed groups capable of forming a reactive intermediate, covalent bonding to DNA and mutagenesis may occur. 83 The molecular graphics views of the benz[a]anthracene diols suggest that in Orientation I there may be ex-

392

Steroids, 1995, vol. 60, M a y

posed portions of the ligand outside the area corresponding to the BCD-bay region of the steroid. Conclusions

The structural basis for the differential estrogen receptor binding affinity of methyl-substituted benz[a]anthracene diols has been investigated. In this series of compounds, the structural feature that decreased the planarity of the polycyclic ring system (bay region methyl substitution) also decreased estrogen receptor binding affinity. This is contrary to many ER ligand systems, in which compounds with the flatter molecular geometry possess lower binding affinity. This may be due to the contribution of dispersive interactions with the receptor to the binding affinity of this class of compounds. Molecular graphics (excluded volume analysis) was used to analyze the fit of these four compounds with a receptor-excluded volume map for the ER. These studies indicate that that these compounds bind to the ER in an orientation such that the anthracene fragment acts as the steroid AB-ring mimic. This result is different from that obtained in previous simpler molecular overlap studies, in which the phenanthrene component of the benz[a]anthracene system was proposed to roughly correspond to the steroidal ABC-ring system. Molecular orbital (AM1) calculations indicated that the 7-methyl substituted compound has the greatest charge similarity to estradiol of the four compounds studied, and that some of the benz[a]anthracene monophenols and diphenols have sufficiently low ionization

Benz[a]anthracene diols as estrogen receptor ligands: Anstead and Kym potential to act as carcinogens by a radical cation process. A linear regression method was used to correct the calculated ionization potentials, using experimental values obtained by two techniques on an analogous set of compounds. It is proposed that the antiestrogenic effects of the benz[a]anthracene diols may be mediated through binding to the aromatic hydrocarbon receptor and that carcinogenesis may be due to metabolic activation of the receptor-bound ligand in the DNA-receptor complex.

16. 17. 18. 19. 20.

Acknowledgments We thank Charles Morreal of Roswell Park Memorial Institute for supplying us with samples of the benz[a]anthracenes that stimulated our interest in these compounds, and John Katzenellenbogen of the University of Illinois for the use of the SYBYL system. This study was funded by the National Institutes of Health (PHS RO1 DK15556). We thank Vickie Watson and Krista Gallagher for preparation of the manuscript.

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