Bioorganic & Medicinal Chemistry Letters 22 (2012) 3713–3717

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Bioorganic & Medicinal Chemistry Letters

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BC-spiro-estradiols. Synthesis and estrogen receptor binding affinity of fournew estradiol isomers

Muhammad Asim a, Daria Klonowska a, Christine Choueiri a, Ilia Korobkov a, Kathryn E. Carlson b,John A. Katzenellenbogen b, Tony Durst a,⇑a Department of Chemistry, University of Ottawa, Ottawa, Canada K1N 6N5b Department of Chemistry, University of Illinois, Urbana, IL 6180, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 4 March 2012Revised 2 April 2012Accepted 4 April 2012Available online 11 April 2012

Keywords:EstradiolBC-spiro-estrogenER binding affinitySynthesis

0960-894X/$ - see front matter � 2012 Published byhttp://dx.doi.org/10.1016/j.bmcl.2012.04.022

⇑ Corresponding author. Tel.: +1 613 562 5800x607E-mail address: tdurst@uottawa.ca (T. Durst).

The synthesis of four new isomers of estradiol in which the ring A to ring C planes are perpendicular toeach other as a result of a spiro BC ring junction is described. Heterocyclic analogs and carbocyclichomologs of these compounds are also reported. Estrogen receptor binding studies show that the spirocompounds with the natural stereochemistry at C9 bind almost as strongly as estradiol but with greaterb to a selectivity. These studies show that the estrogen receptors can readily accommodate isomers ofestrogen with substantially different fixed shapes than the native ligand

� 2012 Published by Elsevier Ltd.

A large number of analogs of estradiol, the natural ligand for theestrogen receptor (ER), have been prepared and evaluated for theirbinding to the ER, and more recently, to the two ER subtypes, ERaand ERb. These analogs of estradiol provide interesting probes ofthe ligand binding pocket of ERa and ERb, and some have potentialfor estrogen pharmaceutical applications. This work was reviewedin detail some time ago,1,2 and the available information showedclearly that both ERa and ERb can accommodate quite readily addi-tional substituents on the steroidal structure, in particular on theb-face at C11 and on the a-face at C7 and C16. All of these deriva-tives are based on the natural, essentially planar ABCD ring archi-tecture of estradiol (1). Substantial additional work describingmany non-steroidal compounds SERMs also supports the conclu-sion that the estrogen receptors are capable of accommodating agreat variety of structures with a varied core composition andgeometries, and peripheral substituents.1–3

We report herein four new isomers of estradiol, 2–5, in whichthe plane of ring A is constitutionally constrained to be perpendic-ular to that of ring C through a spiro BC ring junction (Fig. 1). To thebest of our knowledge, these compounds represent the first iso-mers of estradiol that have been reported in which rings B and Care connected via a spiro-ring as opposed to the usual fused-ringjunction, although certain spiro-nonsteroidal compounds areknown to bind to ER,4,5 to potentiate estrogen action,6 or to inhibitstereoid reductases.7 Since the CD portion of these compounds was

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prepared in enantiomerically pure form,3 all the compounds de-scribed herein were prepared as single enantiomers. Spiro deriva-tive 2, or BC-spiro-estradiol, is most closely related to estradiolsince it is a constitutional isomer that has the same configurationas estradiol at all of its chiral centers. BC-spiro-CD-cis-estradiol,3, also has the same configuration as estradiol at all of its chiralcenters, with the exception that the CD ring junction is cis com-pared to trans in estradiol. The spiro estradiol isomers 4 and 5 havethe non-natural configuration at the spiro carbon designated as C9to allow for a facile comparison with estradiol. In the case of 4, thisresults in a bent shaped structure which was confirmed by an X-ray crystal structure (Fig. 3). Compound 5 can exist in two confor-mations since the cis CD junction allows for a ring C chair-chairinterconversion; the energetically preferred conformation is asshown.

We have also prepared compounds 6 and 7 which are homologsof the spiro estradiols having a six-membered ring B. With the Bring enlarged, the preferred dihedral angle made by the planes ofring A and C is in the 60–70� range. The availability of these iso-meric estradiols with fixed conformations different from estradiol(2–5), the homologs 6 and 7, and the A-CD steroids, structures, 8and 9, previously reported by us,8,9 which can adopt a variety ofconformations due to rotation about the ring A to ring C bond,has allowed us to probe further into how variations in the shapeof ligands closely related to estradiol can affect their binding affin-ity for the estrogen receptors, ERa and ERb.

In a previous paper,9 we described modeling studies which indi-cated that when bound to the estrogen receptors, the A-CD ligands

CH3OH

1, estradiolA to C dihedral angle ~10°RBAα = 100RBAβ = 100

AB

CH3OH

C

HO

B

A

CH3

C

HO

B

AOH

2, BC-spiro-estradiolA to C dihedral angle = 90°RBAα = 0.98 ± 0.26RBAβ = 2.43 ± 0.71

3, BC-spiro-CD-cis-estradiolA to C dihedral angle = 90°RBAα = 6.4 ± 1.7RBAβ = 40.5 ± 10

CH3OH

CB

4, BC-spiro-C9 invertedA to C dihedral angle = 90°RBAα = 0.12 ± 0.03RBAβ = 0.23 ± 0.03

HO

9 CH3C

HO

B

A

5, BC-spiro-CD-cis-C9 invertedA to C dihedral angle = 90°RBAα = 0.34 ± 0.09RBAβ = 0.37 ± 0.09

OHH

9

CH3

COH

HO

6, homo-BC-CD-cis-estradiolA to C dihedral angle ~60-70°RBAα = 3.7 ± 0.97RBAβ = 39.7 ± 1.4

B

A

A

H

HO

1

3

9 16C DD D

DDD

CH3

7, homo-BC-CD-cis-C9-invertedA to C dihedral angle = 60-70°RBAα = 0.05 ± 0.001RBAβ = 0.16 ± 0.02

OHH

9

HO CH3

OH

8, A-CD-transA to C dihedral angle; variableRBAα = 2.38 ± 0.19RBAβ = 9.97 ± 1.3

HO CH3

9, A-CD-cisA to C dihedral angle; variableRBAα = 1.5 ± 0.26RBAβ = 21.5 ± 4.6

HO

OHA A

HH H

Figure 1. Structures of estradiol (1), BC spiro estrogens (2–7), and A-CD estrogens (8, 9). Relative binding affinity (RBA) values are given for ERa (RBAa) and ERb (RBAb). TheRBA value is the binding affinity relative to that of estradiol (RBA = 100), and are determined by a competitive radiometric binding assay using human, full length ERa and ERband [3H]estradiol as tracer, as we have described.10 Values represent the mean ± standard deviation from 2–3 determinations. Ki values can be determined from the RBAvalues (Kcompound

i = RBA � Kestradiold /100; Kd = 0.2 nM (ERa); 0.5 nM (ERb)).

13A to C dihedral angle= 90°RBAa = 0.014 ± 0.001RBAb= 0.020 ± 0.006

CH3

OHH

O

HOCH3

OHH

HO

12A to C dihedral angle= 90°RBAa = 0.061± 0.002RBAb = 0.59± 0.05

H

CH3

OH

O

HO

10, X=H, H; 14, X= ORing A to C dihedral angle= 90°RBAa = 2.46 ± 0.11 RBAa = 1.98 ± 0.40RBAb = 9.05 ± 2.40 RBAb = 6.07 ± 0.77

CH3

OH

O

HO

11A to C dihedral angle = 90°RBAa = 0.31 ± 0.002RBAb = 3.47 ± 0.93

X

Figure 2. Structures of other spiro estrogens (10, 11, 13, 14) and an A-CD estrogen (12). See legend to Figure 1.

3714 M. Asim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3713–3717

8 and 9 adjusted their conformation such that the dihedral anglemade by the aromatic and C rings was approximately 30�. Duringthe course of these studies, we realized that we could probe moredeeply into the shape of ligands capable of binding to the estrogenreceptors by preparing isomers of estrogen with fixed, or nearlyfixed, dihedral angles between these two planes by adding atwo-carbon bridge that created spiro ring compounds. We havedetermined that 2 and 3, which are constitutional isomers of estra-diol with a fixed dihedral angle of 90�, bind to ERb with a relativebinding affinity (RBA) value of 2.4 and 40.5 (RBA estradiol = 100)and to ERa, with an RBA of 1.0 and 6.4, respectively. For compari-son, the conformationally mobile structures 8 and 9 gave RBAs of10 and 21.5 for ERb, and 2.4 and 1.5 for ERa, respectively. The 6-oxo analog of 3, compound 10, also shows significant binding toboth estrogen receptors (Fig. 2). Thus, the quite rigid spiro estro-gens have binding affinities comparable to those of the A-CDligands.

The homo analog 6, in which the ring B is expanded to a sixmembered-ring, has a somewhat more flexible conformation. Nev-ertheless, the A to C dihedral angle is restricted to a range no lessthan about 60–70�. The binding of this compound to the ERs is

comparable to that of 3, while that of the oxa analog 11, with a60–70� dihedral angle, is similar to 10, where the planes are at90�. Interestingly, in contrast to estradiol, which binds at compara-ble levels to ERa and ERb, these compounds show affinity prefer-ence for ERb in the 6 to 15-fold range.

We conclude that the estrogen receptors accommodate almostequally well a series of isomeric or very similar ligands with dis-tinctly different shapes: (a) the natural hormone estradiol, whichis essentially planar, (b) the spiro compounds 2 and 3, structuralisomers of estradiol in which the planes of the aromatic ring andthat of ring C are fixed at 90� to each other, and (c) the homolog8, where the two planes are fixed at 60–70�. These findings areconsistent with the well-appreciated capacity of the estrogenreceptors to bind with good affinity to ligands having structurallydiverse core structures,11,12 as well as emerging evidence for the‘plasticity’ of the ligand binding pocket13 and the concept thatthe final shape of this pocket is only determined during ligandbinding, a process that enables the lower portion of the ligandbinding domain to complete its folding.3

During the preparation of the above compounds, we also iso-lated the spiro estrogen isomers with the opposite, non-natural

Figure 3. ORTEP of compound 4 and compound 26.

M. Asim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3713–3717 3715

estrogen configuration at the spiro carbon. These compounds, 4, 5,7 and 13, show considerably reduced binding to ERa and ERb rela-tive to compounds 2, 3 and 6 but comparable to the A-CD com-pound 12 having the non-natural configuration at C9 (Figs. 1 and2). The reduced binding affinities of these compounds comparedto the analogs with the natural configuration at C9 is expectedsince the relationship of the hydroxyl groups, the key pharmaco-phore groups, in these compounds, especially in 4, is considerablyaltered relative to estradiol and to the higher affinity ligands, 2, 3,6, 11 and 14.

The synthesis of the spiro estrogens was relatively straightfor-ward, with the exception of spiro-estradiol, 2, which has the sameabsolute configuration at all of its chiral carbons as estradiol (itssynthesis is described later). The preparation of the spiro deriva-tives 3 and 5 commenced with the addition of 2-(30-methoxy-phenyl)-1-ethanemagnesium bromide 15 to the saturated CDring ketone 16,14 affording adduct 17, which was cyclized to a mix-ture of the spiro derivatives 18 and 19 upon exposure to MeSO3Hin nitromethane at �10 �C. These compounds were separated viapreparative HPLC. Reaction with NaSC2H5 in DMF at 160 �C for10 min afforded in essentially quantitative yield compounds 3and 5,15 respectively (Scheme 1).

The spiro-6 analogs 6 and 7 were also prepared following thesequence shown in Scheme 1 starting with the addition of thehomolog of 15, namely 3-(30methoxyphenyl)-1-propanemagne-

MeO MgBr

OMOM

H

HO

H3CO15 17

a

Scheme 1. Reagents and conditions: (a) compound 16 (91%); (b) C

sium bromide with the cis-ketone 16. When applied to the transfused CD-ketone 20,16 this same sequence gave an excellent yieldof the initial adduct 21. Acid-catalyzed cyclization gave only thespiro derivative 22 resulting from attack of the aromatic ring onthe carbocation intermediate from the sterically less hindered bot-tom side, opposite the methyl group. Demethylation afforded 4,whose structure was secured by X-ray structure (Fig. 3) determina-tion (Scheme 2).

Since the acid-catalyzed cyclization-deprotection sequencestarting with 21 resulted only in the isomer 4, having the unnaturalconfiguration at C9, an alternate approach to the estradiol isomer2, which has the trans CD ring fusion and thus most closely resem-bles estradiol was required. As shown in Scheme 3, addition of thespecies 15 to the unsaturated enone 2316 afforded the expectedproduct 24. Acid-catalyzed dehydration gave, somewhat surpris-ingly, the D14,15 alkene 25 as a single diastereomer, which was re-acted with mCPBA to give exclusively the epoxide 26, whosestructure was secured by X-ray crystallography (Fig. 3). This notonly verified the configuration at C9 relative to the substituentsin ring D but also showed that the C ring existed in the chair con-formation. Acid-catalyzed rearrangement of the epoxide resultedin a hydride shift to form the ketone 27, which has the trans CDring junction, as again verified by an X-ray crystal structure.

The conversion of 27 into 2 was accomplished by the followingsequence: NaBH4 reduction gave a mixture of alcohols, with themajor compound resulting from the introduction of hydride fromthe same side as the angular methyl group; the C–H of this alcoholshowed coupling of 11 Hz coupled with the adjacent axial C-14methine hydrogen. Conversion of this alcohol to its thiocarbonate28 was quite troublesome and occurred in less than 20% yielddue to its highly hindered nature. Finally, reaction of 28 withBu3SnH catalyzed by AIBN and subsequent removal of both themethoxy and acetate groups gave the desired 2. The deoxygenationof an alcohol by the above sequence has been described numeroustimes as a preferred method for converting an alcohol into the cor-responding hydrocarbon.17

The synthesis of the oxa spiro compounds followed ourprevious syntheses of the A-CD compounds (Scheme 4). The lithioderivative derived from di-MOM protected 3-hydroxymethyl-4-bromophenol, 29, was added with the cis-CD moiety 16. Themixture of benzylic alcohols thus obtained was treated with TsOHin methanol to afford a separable mixture of 10 and 13. A similarsequence when applied to 30 gave 11 and the homolog of 13;the ER binding of the latter compound was not determined(Scheme 4).

The preparation of the spiro lactone 14 involved metallation ofthe bromo amide 31 and coupling with 16 to afford a mixture ofthe adducts 17. Acid catalyzed hydrolysis of 17 afforded a mixtureof spiro lactones from which pure 14 was obtained via preparativeHPLC (Scheme 5).

The present study represents the first systematic probe of theestrogen receptors using isomers and simple analogs of estradiolhaving fixed shapes very different from that of the fused and planartetracyclic steroid. The spiro compounds 2, 3, 6, 10, 12 and 14,

OH

HH3CO +

OH

H

OCH3

3 5

18 19

b

c c

H3SO3H/CH3NO2/�10 �C (85%); (c) NaSC2H5/DMF/160 �C (95%).

OTBS

O

H3C OTBS

HO

H3CO

CH3

H

HO OH

a

CH3

OH

OAcCH3

H

OAcH3CO H3CO

2

23 24

b, c

CH3OAcH3CO

e f , g h, i

9

9

25

27O

SOPh28

CH3OAcH3CO

926

Od

Scheme 3. Reagents and conditions: (a) compound 15 (90%); (b) CH3SO3H/CH3NO2/�10 �C (80%); (c) Ac2O/py(99%); (d) mCPBA/DCM (82%); (e) BF3Et2O/DCM (88%); (f)NaBH4/CH3OH (96%); (g) Ph-O-C(S)-Cl/DMAP (18%); (h) AIBN/Bu3SnH (82%); (i) NaSC2H5/DMF/160 �C (97%).

OTBS

HO

OTBS

H

HO

H3CO

OH

H

H3C

H

OCH3

OH

HO20 21422

a b c

Scheme 2. Reagents and conditions: (a) compound 15 (90%); (b) CH3SO3H/CH3NO2/�10 �C (80%); (c) NaSC2H5/DMF/160 �C (93%).

MOMO OMOM

Br

a, b, c

CH3OH

O

HO

O

CH3

OH

HO+( )n

( )n( )n10, n = 1

11, n = 2 13, n = 129, n = 130, n = 2

Scheme 4. Reagents and conditions: (a) nBuLi/THF/�78 �C; (b) compound 16 (15% & 14% n = 1 & n = 2); (c) H(+).

MOMO

Bra, b

CH3

OH

O

HO

14

NEt2

OO

OMOM

HO

MOMO

c, d

31 17C(O)NEt2

Scheme 5. Reagents and conditions: (a) nBuLi/THF/�78 �C; (b) compound 16 (30%); (c) H(+) (97%); (d) HPLC spearation.

3716 M. Asim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3713–3717

which have the natural estrogen configuration at C9, show goodbinding affinity when compared to estradiol, but unlike estradiolthey exhibit modest to good selectivity (3 to 14-fold) for ERb overERa. These results verify the previous hypotheses that the overallshape of the estrogen molecule can be changed quite substantiallywithout significantly lowering the binding affinity of such mole-cules to the estrogen receptors, and they are also consistent withevidence that the ligand binding pocket is plastic and develops adefined shape only upon ligand binding. The spiro compounds withinverted configuration at C9 relative to estradiol, on the otherhand, show much weaker binding affinity. These results should en-able us to model the estrogen receptor–ligand interactions withgreater confidence and allow us to generate additional compoundswith more predictable and potentially stronger binding to theestrogen receptors.

Acknowledgments

The support of this project by the Canadian Breast Cancer Foun-dation and NSERC Canada [Discovery Grant 1169 to T.D.] and the

United States National Institutes of Health [PHS R01DK015556 toJ.A.K.] is gratefully acknowledged. C.C. would like to thank Dr.Vijayaratnam Santhakumar for helpful discussions during herinternship at Astra Zeneca in Montreal.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2012.04.022.

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