Determination of the Absolute Configuration of Aromataseand Dual Aromatase-Sulfatase Inhibitors by Vibrationaland Electronic Circular Dichroism Spectra Analysis

SERGIO ABBATE,1,2* GIOVANNA LONGHI,1,2 ETTORE CASTIGLIONI,1,3 FRANCE LEBON,1,2 PAUL M. WOOD,4

LAWRENCE W. L. WOO,4 AND BARRY V. L. POTTER4

1Dipartimento di Scienze Biomediche e Biotecnologie, Universita di Brescia, Brescia, Italy2Consorzio Interuniversitario Scienze Fisiche Della Materia, CNISM, Roma, Italy

3JASCO Corporation, 2967-5 Ishikawa-Cho, Hachioji-Shi, Tokyo, Japan4Department of Pharmacy and Pharmacology and Sterix Ltd, Medicinal Chemistry, University of Bath,

Claverton Down, Bath, United Kingdom

ABSTRACT The absolute configuration of a newly designed, letrozole-based chiralaromatase inhibitor that could not be defined by crystallographic techniques has beendetermined by means of vibrational and electronic circular dichroism and by opticalrotation measurements combined with density functional theory calculations on possibleconformers. The same absolute configurational assignment can be applied to the indi-vidual enantiomeric sulfamate esters, which are derived from the corresponding enan-tiomers of the chirally separated parent phenols, based on the similarity of the ECDspectrum of the sulfamate derivative to that of its phenolic precursor. The two enantio-meric sulfamate esters studied here are the first examples of nonsteroidal dual aroma-tase-sulfatase inhibitor whose activities have been evaluated on optically resolved enan-tiomers. Chirality 21:802–808, 2009. VVC 2009 Wiley-Liss, Inc.

KEY WORDS: vibrational circular dichroism; electronic circular dichroism; opticalrotation; DFT calculations; aromatase inhibitors; dual aromatase-sulfatase inhibitors; letrozole

INTRODUCTION

A range of aromatase inhibitors (AIs) have been suc-cessfully developed for the treatment of hormone-depend-ent breast cancer.1 These compounds inhibit the aroma-tase enzyme which is responsible for the conversion ofandrogens to estrogens thus depriving the tumor of thesegrowth promoting steroids. However, a new strategy forestrogen deprivation has arisen from the development ofinhibitors of the enzyme steroid sulfatase (STS) whichconverts notably estrone sulfate to its biologically activeunsulfated form, i.e., estrone. Recently, dual aromatase-ste-roid sulfatase inhibitors (DASIs) have been developedwith dual inhibition being achieved by incorporation of thepharmacophore for STS inhibition into established AIssuch as letrozole, anastrozole, and YM511.2–4

The compounds considered here are derivatives of thesymmetric AI letrozole which comprises of two para-cy-anophenyl rings, and a triazole ring which is a motif pres-ent in a number of nonsteroidal AIs. As part of the devel-opment of letrozole-based DASIs, a series of chiral com-pounds were prepared by replacing one of the cyanogroups present in letrozole with a sulfamate group, with orwithout additional substituent on the same phenyl ring.

In this work, we determine the absolute configuration(AC) of the two enantiomers of a brominated phenolicletrozole derivative (I, Scheme 1) which is a precursor to

the final corresponding sulfamoylated compound (II,Scheme 1). The two compounds I and II were preparedand the racemic mixture of compound (I) was separatedby semi-prep chiral HPLC with a Chiralpak1 AD-H columnas previously described.5 The optical rotation (OR) meas-ured in ethanolic solutions for (1)-I is [a]20D 5 184.38(c3.9), and for its antipode (2)-I is [a]20D 5 279.18(c 3.6).The conversion of individual phenolic enantiomer to thecorresponding sulfamate was verified to occur without rac-emization by chiral HPLC as shown by the retention ofenantiomeric purity. As ethanolic solutions, the enantiom-ers of p-sulfamate (II) have optical rotations of [a]20D 5

134.28(c 0.97) for (1)-II, and [a]20D 5 230.78(c 0.95) for(2)-II.

Each phenolic enantiomer was tested against aromataseand in addition to this each enantiomer of the correspond-ing sulfamate was tested against both aromatase and STS.The enantiomers were found to have differing activities

*Correspondence to: Sergio Abbate, Dipartimento di Scienze Biomedichee Biotecnologie, Universita di Brescia, Viale Europa 11, 25133 Brescia,Italy. E-mail: abbate@med.unibs.it

Contract grant sponsor: Italian Ministry of University and Research.

Received for publication 11 July 2008; Accepted 3 November 2008DOI: 10.1002/chir.20685Published online 22 January 2009 in Wiley InterScience(www.interscience.wiley.com).

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VVC 2009 Wiley-Liss, Inc.

against each enzyme and (1) enantiomer of the phenolwas similar in potency against aromatase to letrozole.5

After the HPLC separation, it was necessary to deter-mine the AC of each enantiomer of I. Unlike the racemicmixture, the separated enantiomers could not be crystal-lized successfully, as slow evaporation of the solvent froma solution of either enantiomer gave an oil. For this rea-son, it was necessary to use a combination of chiropticalspectroscopic techniques for the determination of AC:vibrational circular dichroism (VCD)6–10 and electronic cir-cular dichroism (ECD)11 in conjunction with DFT calcula-tions. Herein we reconsider the work that was previouslypresented in Ref. 5 with the use of more accurate calcula-tions and further experiments.

METHODS

VCD measurements on compound I were conducted witha JASCO FVS4000 apparatus equipped with a MCT detec-tor, using a BaF2 cell of 50 lm pathlength, 4 cm21 resolu-tion, and 4000 scans. Both eluted enantiomers were dis-solved in CDCl3, 0.26 M for (1)-I and 0.23 M for (2)-I. Agood mirror-image condition was obtained for the twoenantiomers; however, to eliminate baseline drift we pres-ent for compound (1)-I the half difference, i.e., (1/2)[(1)-I 2 (2)-I].

ECD measurements were taken at room temperature, ona JASCO 815-SE spectropolarimeter, flushed with drynitrogen using 0.1 cm and 0.01 cm quartz cuvettes adopt-ing the following conditions: 100 nm/min scanning rate,2 nm resolution, and 10–20 scans. Measurements weretaken on both enantiomers of compounds I and II in meth-anolic solutions at 1023 M concentration and spectra weresolvent subtracted. Measurements were also taken in chlo-roform (limited to the range 233–350 nm); they gaveessentially identical results (the data are available onrequest).

Calculations

The geometries of eight stable conformers of compoundI were optimized at B3LYP/6-31G* and B3LYP/TZVPlevel. AC can be determined by comparing experimentalVCD spectra with the spectrum calculated for one chosenconfiguration (R in this case): B3LYP/TZVP based DFTcalculations have been performed on the correspondingoptimized geometries, harmonic rotational strengths havebeen calculated using gauge invariant atomic orbitals(GIAO) as implemented in Gaussian03 package.12 The cal-culated harmonic frequencies, dipole, and rotational

strengths were used to generate Lorentzian bands withhalf width 4 cm21. The final calculated spectrum is aweighted average of spectra obtained for the eight confor-mations taking into account Boltzmann population factors,as obtained from calculated free energies.

The ECD spectrum of the R configuration of compoundI was calculated using a TDDFT approach11 at the samefunctional and basis set level B3LYP/TZVP, consideringthe same conformers and the same population factorsemployed for VCD. The rotational strength calculationshave been carried out both in the velocity and length for-malism using the Gaussian03 package.12 The two resultsare almost identical; 50 singly excited states have beenconsidered. From calculated dipole and rotationalstrengths, extinction coefficients e and De can be eval-uated.11,13 We have assumed that each electronic transi-tion gives rise to a Gaussian band; a width of 9 nm gives agood fitting with experimental data.

In addition, the specific optical rotation OR at the so-dium D line has been calculated using TDDFT B3LYP/6-31G* for the eight conformers.

RESULTS AND DISCUSSIONConformational Aspects

Before simulating the CD spectra, the possible confor-mations of a given configuration (vide infra) of I wereexamined. This resulted in the discovery of eight most sta-ble conformations each one optimized at B3LYP/6-31G*level in vacuo. The same conformers have been subse-quently optimized using a TZPV basis set. The moleculecontains an asymmetric carbon and despite the lack of ann-fold axis of symmetry it is propeller-like.14,15 As docu-mented in Table 1, the three blades of the propeller aretwisted with respect to the central methine C*H, thus giv-ing rise to a right handed or a left handed propeller; thethree aromatic rings are not inclined in the same way. Forthis reason we classify four conformations as pseudo-D,and the other four as pseudo-L (see Scheme 2 below).14

The calculations show that the pseudo-L conformers aremore stable than pseudo-D conformers (with an exceptionconsidering B3LYP/TZVP).

The geometries calculated with the two basis sets arequite similar: for this reason we just report the dihedralangles obtained with the more extended basis set. Com-parison of the eight conformations reveals that in somecases two of the rings are nearly parallel with respect tomethine (within 10–208) and the third ring is at about 35–458 (conformers 1 and 2 for pseudo-L, and 2 and 3 for

Scheme 1. Definition of Phenolic (I) and sulfamoylated (II) compounds related to letrozole.

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pseudo-D), whereas in other cases, the three rings are ori-ented more similarly to one another (in Table 1 see thecalculated values for the dihedral angles). Displayed inFigure 1 are the eight most populated conformers, repre-sented in a ball and stick format.

As a further check, a conformational search was con-ducted based on semiempirical method PM3: in additionto the conformations reported in the table, lower energygeometries were found, in which the three rings arenearly parallel to the methine: precisely four cases thatcorrespond to the two orientations of the phenolic ring,with the bromine atom either on the same side or oppositeto the methine, and analogously for the two orientationsof the triazole ring. All these initial conditions, optimizedat the B3LYP/6-31G* level, converged to cases reportedin Table 1. Finally we checked that the conformations withthe O��H bond pointing away from the Bromine atomthey have energies 3–4 kcal/mol higher than the onesconsidered here.

Assignment of the Absolute Configuration

VCD spectra. We report in Figure 2 the experimentaland calculated mid-IR VCD and absorption spectra forcompound I. The R configuration was chosen for the calcu-lations and the eight conformations were fully optimizedand were verified to be real energy minima. Free energyvalues were calculated for each conformation to utilizeBoltzmann statistics to evaluate the population of each con-former at room temperature. In the same Figure 2, wecompare the experimental spectra to the calculated spectrawith the two basis sets: the latter have been Boltzmannweighted averaged over conformers, and in the case ofTZVP basis set we present also the mere arithmeticaverage.

All three calculated averages match fairly well experi-mental absorption and VCD spectra of the (1)-I com-pound. For this reason, this compound is attributed the Rconfiguration. In many previously reported examples,VCD had been used to determine the AC of a mole-cule16,17; also in this case VCD permits the unambiguousassignment of the AC of (1)-I as R. For the sake of com-pleteness, we report in Figure 3 the absorption and VCDspectra of the eight conformers calculated at B3LYP/TZVP level. One may notice that the sign of the bands in

the VCD spectra is very sensitive to rotation of a phenylring and/or to ‘‘helicity’’ change whereas the absorptionIR spectra are quite similar for all eight conformers.

A more detailed analysis of the comparison between cal-culated and observed VCD spectra (see Fig. 2) revealsthat with the first basis set, already used in Ref. 5, two neg-ative bands are calculated and not observed at 1370 cm21

and at 1435 cm21. For this reason, to obtain a better agree-ment, we considered here B3LYP/TZVP: a good corre-spondence is observed for the 1141 (1) 1170 (2) 1182(sh 1) 1189 (1) 1206 (2) cm21 bands (in correspondencewith the last wavenumber we predict a more structuredfeature) and for 1248 (1) 1265 (1) 1309 (1) 1334 (1)1412 (1) cm21 bands (in the last case we predict also anegative not observed feature) 1429 (1). The new calcula-tions further confirm our previous configuration assign-ment.

Because of the presence of many conformations, adirect evaluation of the populations from experimentaldata is not possible, for this reason we propose in Figure 2also a comparison with the arithmetic average of the calcu-lated spectra, which appears in quite good correspondencealso around 1200 cm21.

ECD spectra. In Figure 4, we report the comparisonof the experimental and calculated UV and ECD spectra ofcompound I. The calculated spectrum presented in Figure4 is obtained by TDDFT calculations at B3LYP/TZVPlevel, rotational strength in the velocity representation,averaging over the spectra corresponding to the eight con-formations considered above. The ECD experimental spec-trum is quite rich in bands and is well reproduced bysimulated spectra despite the fact that the level of calculusis not very high and only pure electronic transitions in

TABLE 1. Geometry parameters s1, s2, and s3 (see Scheme 2 for definition; s2 is defined considering methine and the CCbond adjacent Br, s3 considering methine and the NC bond), Gibbs free energy values and percent population factors of

eight stable conformers, specific optical rotation at the sodium D line as calculated by B3LYP/6-31G*

s1 s2 (TZVP) s3DG cal/mol(6-31G*)

pop-DG(6-31G*) [a]D(6-31G*)

DG cal/mol(TZVP)

pop-DG(TZVP)

1 pseudo-L 2138.0 2165.7 9.9 0.0 22.9% 343.7 0.0 21.1%2 pseudo-L 2140.6 14.0 9.8 71.5 20.3% 529.2 420.4 10.2%3 pseudo-L 2136.7 2147.9 2144.1 115.5 18.8% 154.1 47.1 19.4%4 pseudo-L 2135.4 30.2 2144.0 305.0 13.6% 250.1 189.5 15.2%1 pseudo-D 142.9 140.4 148.0 690.9 7.0% 2390.9 718.5 6.1%2 pseudo-D 163.3 236.0 211.5 705.3 6.8% 2322.9 171.9 15.7%3 pseudo-D 160.3 146.2 210.1 712.2 6.7% 2649.0 690.9 6.4%4 pseudo-D 143.5 239.3 147.3 1041.7 3.8% 284.3 746.1 5.8%

Scheme 2. Definition of pseudo-L and pseudo-D conformations for I.

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vacuo are considered. The good prediction of a quite struc-tured spectrum in this example makes the ECD spectrumuseful in assigning AC. Indeed, in a case like that cur-rently considered where many conformers are populatedand there are bands of both signs, it is important that bothVCD and ECD data allow one to arrive at the same conclu-sion for AC. The calculated ECD spectra for each of theeight conformations along with those obtained experimen-

tally are shown in Figure 5. In all of the D conformers,negative bands are observed at high wavelength while thepseudo-L conformers present the first transitions at lowerwavelength and the corresponding ECD bands have oppo-site sign. For these reasons, the ECD spectrum at highwavelength (� 290 nm) is determined by pseudo-D con-formers despite their low population factors. Reproductionof the ECD spectrum is strongly dependent not only on

Fig. 2. VCD and IR absorption spectra. Bottom trace: (black, bold face) experimental spectrum of (1)-I (see text). Top trace: calculated spectrum atB3LYP/6-31G* level for the R configuration of I (weighted average taking into account populations obtained by calculated Gibbs free energies).5 Calcu-lated frequencies have been shifted by 30 cm21. Middle upper trace: calculated spectrum at B3LYP/TZVP level for the R configuration of I (weightedaverage taking into account populations obtained by calculated Gibbs free energies), middle lower trace: calculated spectrum at B3LYP/TZVP level forthe R configuration of I (arithmetic average), calculated frequencies have been shifted by 20 cm21. [Color figure can be viewed in the online issue,which is available at www.interscience.wiley.com.]

Fig. 1. Ball and stick representation of the most populated conformers of molecule I (see Scheme 1 and text). [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]

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the calculated rotational strengths but also on the chosenbandwidth. The assumption of 9 nm bandwidth for all cal-culated features gives acceptable ECD and absorptionintensities, as seen from Figure 4. We have verified thatusing a 0.2 eV bandwidth instead11 does not drasticallychange the result.

In Figure 6, we report the experimental ECD spectra forcompound II. Comparing the 190–240 nm region for thephenolic and the sulfamoylated compounds reveals thatboth exhibit the same behavior. For the (1) compoundsthere are a negative band (at 195 nm) and two positiveones (at 210 and 219 nm). The corresponding bands withthe opposite sign are observed for the (2) compounds. Inthe 240–340 nm region, the phenolic compound (1)-Ipresents weak negative CD features at 270, 278, 284, and291 nm that reduce to two features for the sulfamoylatedcompound at 271 and 279 nm. Based on this comparison,we may safely assign the AC of compound II as R-(1) andS-(2). A chiral HPLC study of the two enantiomers of IIfollowing sulfamoylation confirmed that no racemizationhad taken place during this step.5 We can thus safely con-clude that the sulfamate retains the AC of its correspond-ing phenol, as confirmed by the observation that the ECDspectra of the two compounds are quite similar. In othercases presented in the literature, the AC of a compoundhas been assessed by the preparation of a convenient de-rivative as long as the synthesis of the derivative leavesthe stereogenic carbons unchanged (see for example18).

Optical rotation. Finally, we have compared the opti-cal rotation data of I with the results obtained fromTDDFT calculations. Ab initio methods for calculating ORwere developed in 199719,20 and TDDFT methods forcalculating OR21 were subsequently implemented in

Fig. 3. VCD and IR absorption spectra. Bottom trace: (black, bold face) experimental spectrum of (1)-I. Second trace: calculated spectra at B3LYP/TZVP level (weighted average taking into account populations obtained by calculated Gibbs free energies). Black weaker traces: calculated spectra atB3LYP/TZVP level for each conformer in order of decreasing population. Calculated frequencies have been shifted by 20 cm21. [Color figure can beviewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 4. ECD spectra. Bold dark blue trace: experimental spectrum ofcompound (1)-I. Bold light blue trace: experimental spectrum of com-pound (2)-I. Black trace: calculated spectrum (B3LYP/TZVP) for R con-figuration (average over eight conformers with calculated population fac-tors). The spectra were recorded on 1023 M CH3OH solutions in therange 190–340 nm. Inset A: enlargement of 240–340 nm CD spectrum.Inset B: UV spectra experimental (bold black trace) and calculated (blacktrace). [Color figure can be viewed in the online issue, which is availableat www.interscience.wiley.com.]

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Gaussian. In Table 1 we report the OR calculated at the so-dium D line for the eight conformers of I. A conforma-tional average, obtained from populations based on DG,gives a value of 11538 for the R configuration which com-pares well with the experimental value. A problem in thiscase is that of the eight conformers considered, the fourpseudo-L conformers give positive OR whereas the fourpseudo-D give negative OR. For this reason, this datumper se may not be cogent in determining the AC, further-more it is well known that optical rotation can be safelyused to assess AC only if considered at various wavelength(ORD). However, this case, used in combination with thetwo previously commented spectroscopic techniques, sug-gests a further proof of the AC and of the prevalence ofpseudo-L forms.

CONCLUSIONS

The concerted use of chiroptical spectroscopies, accom-panied by DFT calculations, is an efficacious method todetermine AC. Careful examination of VCD and ECD spec-tra permits AC assignment even when the molecule has

many different conformers. In this particular case, it waspossible to determine the AC of compound I despite thepresence of conformers of opposite helicity, because com-pounds I and II are propeller-like molecules. TDDFT cal-culations were essential to interpret ECD spectra since cal-culations based on exciton interactions like the DeVoemethod would be quite complicated and in any case wouldrequire a precise evaluation of the reciprocal orientation ofthe three aromatic rings.

The observed ECD spectrum of the sulfamate com-pound II is quite similar to that of the phenolic parent,compound I, and thus has allowed us to conclude (in con-junction with the chiral HPLC test reported in Ref. 5) thatthe AC of the parent compound is preserved. This determi-nation of absolute configuration will permit the explorationof potentially important stereochemical factors in the rec-ognition of aromatase- and dual-aromatase sulfatase inhibi-tors by their target proteins.

ACKNOWLEDGMENTS

We thank Sterix, a member of the Ipsen Group, forsupport.

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Fig. 5. ECD spectra. Top traces: calculated ECD spectra for L con-formers of R configuration I-compound. Bottom traces: calculated ECDspectra for D conformers of the same configuration. Bold dark blue trace:experimental spectrum of compound (1)-I. Bold light blue trace: experi-mental spectrum of compound (2)-I. Black trace: calculated spectrum forR configuration for the average over the eight conformers according tocalculated population factors. The experimental and the calculated averagespectra, which are obviously less intense than the single conformationspectra, have been multiplied by 2 as reported in the secondary axis. Cal-culations conducted at B3LYP/TZVP level. [Color figure can be viewed inthe online issue, which is available at www.interscience.wiley.com.]

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Chirality DOI 10.1002/chir

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