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s t e r o i d s 7 2 ( 2 0 0 7 ) 809–818

avai lab le at www.sc iencedi rec t .com

journa l homepage: www.e lsev ier .com/ locate /s tero ids

lucidation of the opening of the epoxidic ring of the�-acetoxy-14�,15�-epoxy-5�-cholest-8-en-7-one byethanol, using NMR techniques assisted by a

onformational study through theoretical calculations

ario Anastasia ∗, Pietro Allevi, Raffaele Colombo, Elios Gianniniipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universita di Milano, via Saldini 50, I-20133 Milano, Italy

r t i c l e i n f o

rticle history:

eceived 3 April 2007

eceived in revised form

6 May 2007

ccepted 6 July 2007

ublished on line 14 July 2007

eywords:

a b s t r a c t

This paper demonstrates that the crystallization of 3�-acetoxy-14�,15�-epoxy-5�-cholest-8-

en-7-one from methanol affords the 3�-acetoxy-9�-methoxy-15�-hydroxycholest-8(14)-en-

7-one. The structure of this steroid, which shows an apparently anomalous UV absorption

maximum, is determined by high field NMR experiments, supporting the coupling constant

values assignments and the NOE contacts by a conformational study through theoretical

calculations at the B3LYP/6-31G* level. The computational study also justifies the observed

UV absorption of the steroid, thus demonstrating the usefulness of computer chemistry in

providing support for the identification of unknown compounds.

xidation

-Chloroperoxybenzoic acid

�-Acetoxy-5�-cholesta-8,14-dien-

-one

© 2007 Elsevier Inc. All rights reserved.

be formed (Scheme 2), as an intermediate allylic carbocation

poxide

. Introduction

ome years ago, while studying the synthesis of oogoniols1] (a group of closely related steroidal sex hormones ofater mold Achlya), we used the oxidation of 3�-acetoxy-5�-

holesta-8,14-dien-7-one (1) with 3-chloroperoxybenzoic acidmCPBA) for the preparation of 3�-acetoxy-14�,15�-epoxy-5�-holest-8-en-7-one (2, Scheme 1)—an useful starting materialor the preparation of the steroidal moiety of oogoniols.

We have previously demonstrated that the dienone 1 iseadily transformed into a mixture of the epoxy ketone 2nd of 3�-acetoxy-15�-hydroxy-5�-cholesta-8(14),9(11)-dien-

-one (3), obtained, after chromatographic separation, in 61%nd 19% yields, respectively. The same reaction was firsteported by Fieser et al. [2] who obtained a different unidenti-

∗ Corresponding author.E-mail address: mario.anastasia@unimi.it (M. Anastasia).

039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.oi:10.1016/j.steroids.2007.07.004

fied product X (isolated in a 45% yield), after crystallization ofthe reaction mixture from methanol. In our previous work [1]we speculated, based on mass spectral evidences, that com-pound X could derive from methanol addition to the epoxidering of 2 during crystallization. However, at that time, we con-cluded that the obtained physico-chemical evidences werenot sufficient to unequivocally establish the structure of thecompound, and the obtained crystals were not suitable for anX-ray crystallographic analysis. As a matter of fact, in spiteof the simple reaction which may occur during crystallizationof the epoxy ketone 2, four different steroids can in principle

may form during the epoxide ring opening. Moreover UV, IR,and NMR techniques did not allow a clear identification of oneof them.

810 s t e r o i d s 7 2 ( 2 0 0 7 ) 809–818

-ace

Scheme 1 – Steroids obtained from oxidation of 3�

In this paper we unequivocally assign the structure 4 tocompound X originally isolated by Fieser et al. [2], on thebasis of the results obtained by recent NMR techniques andof a conformational study trough theoretical calculations atthe B3LYP/6-31G* level. In particular, the computational studyallowed us to drive and facilitate the analysis and the inter-pretation of the high field NMR experiments, supporting thestereochemical assignments made by the evidenced NOE con-tacts and the coupling constant values between the hydrogenatoms of compound 4. Finally, the computational study alsopermitted us to rationalize the apparently anomalous UVabsorption maximum observed for compound 4.

2. Experimental procedures

2.1. General methods

Melting points were determined on a Thomas Hoover capil-lary melting points apparatus and were uncorrected. Infrared(IR) spectra were recorded for solutions in CDCl3 on a Perkin-Elmer 1420 instrument. Ultraviolet (UV) spectra were recordedfor solution in EtOH on a Perkin-Elmer lambda 11/BIO instru-ment. Optical rotations were taken on a Perkin-Elmer 241polarimeter equipped with a 1 diameter tube; [˛]D values aregiven in 10−1 degree cm2 g−1 and the concentration are givenin g/100 mL.

Mass spectra were obtained using a Hewlett Packard 5988Aspectrometer by direct inlet (electron impact, EI) or a Finni-gan LCQdeca (ThermoQuest) ion trap mass spectrometer fittedwith an electrospray source (ESI). The spectra were collected

Scheme 2 – Possible structures of the compound deriv

toxy-5�-cholesta-8,14-dien-7-one (1) with mCPBA.

in continuous flow mode by connecting the infusion pumpdirectly to the ESI source. Solutions of compounds wereinfused at a flow rate of 5 mL/min. The spray voltage was setat 5.0 kV in the positive mode with a capillary temperature of220 ◦C. Full-scan mass spectra were recorded by scanning am/z range of 100–2000.

The progress of all reactions was monitored by thin-layerchromatography (TLC) carried out on 0.25 mm E. Merck sil-ica gel plates (60 F254) using UV light and 50% sulphuric acidfollowed by heating as developing agent. E. Merck 230–400mesh silica gel was used for rapid column chromatography[3].

2.2. Spectroscopy

Nuclear magnetic resonance (NMR) experiments were doneon a Bruker AMX500 spectrometer, operating at 500.13 MHzfor 1H and at 125.76 MHz for 13C and equipped with a 5 mminverse-geometry broadband probe. All the NMR spectra wererecorded at 298 K in a CDCl3 or C6D6 solution and the chemicalshifts are reported on a ı (ppm) scale and are referenced in 1Hspectra to a solvent residue proton signal (ı 7.26 and 7.15 ppmfor CDCl3 and C6D6, respectively) and in the 13C spectra toCDCl3 or C6D6 signal (central line at ı 77.0 and 128.02 ppm,respectively). For 1H and 13C, the 90◦ pulse widths were 9.6and 8.4 �s, respectively.

All data was collected with the pulse sequences and phase-cycling routines provided in the Bruker libraries of pulseprograms. In 2D experiments the acquisition parameters wereas follows.

ed from the addition of methanol to compound 2.

2 0 0

Ni4ttdu

25i

HHiF(i

tp

fsw2lJ

2

Tmtsffdtitttwo0fm3c0TIionmet

s t e r o i d s 7 2 (

For 1H–1H correlation 2D NMR experiments (COSY, TOCSY,OESY): relaxation delay 2.0 s; eight transients in each exper-

ment; spectral width 5.95 ppm (2976 Hz) in both dimensions;K points in t2 and 2 K experiments in t1, linear prediction

o 4 K. For NOESY an acquisition in TPPI mode and a mixingime of 1.0 s (for 0.5 and 1.5 s were not observed significantifferences) were used; for TOCSY a mixing time of 80 ms wassed.

For J-resolved 2D 1H NMR experiments: relaxation delay.0 s; eight transients in each experiment; spectral width.95 ppm (2976 Hz) in F2 and 0.08 ppm (40.0 Hz in F1; 4 K pointsn t2 and 128 experiments in t1, linear prediction to 256.

For 13C–1H correlation 2D NMR experiments (HSQC,MBC)): relaxation delay 2.5 s for HSQC and 1.0 s forMBC; delay for inversion recovery 0.5 s; eight transients

n each experiment; spectral width 5.95 ppm (2976 Hz) in

2; 154.09 ppm (19,379 Hz) in F1 for HSQC and 212.02 ppm26,666 Hz) in F1 for HMBC; 4 K points in t2 and 1 K experimentsn t1, linear prediction to 2 K.

Data processing, including sine-bell apodization, Fourierransformation, phasing, symmetrisation and plotting, wereerformed with the use of the Bruker software packages.

1H chemical shifts were determined from 1D spectra orrom F2 projections of 1H–1H J-resolved 2D experiments. Thetereochemistry of methylene protons of steroidal nucleusas deduced from the splitting patterns of signals or by NOESY

D experiments. 1H–1H coupling constants were derived fromine spacing of resolution-enhanced 1D spectra or from 1H–1H-resolved experiments.

.3. Computational methods

he gas phase conformational searches were performed usingolecular mechanics force field (MM+; implemented within

he HyperChem software package [4]) and varying all theignificant dihedral angles in a Metropolis Monte-Carlo con-ormational search. The search was performed with a rangeor acyclic or ring torsion variation of ±10–120◦. The Ran-om Walk, and Metropolis Criterion use T = 300 K, switchingo 400 K. The maximum allowed atomic displacement usedn the generation of trial configuration was set to 0.5 A andhe number of steps required before equilibration was seto 1000. The iteration in the Monte-Carlo search was seto 20,000, with a limit of 1000 conformers and an energyindow of 10 kcal mol−1. The accepted conformations wereptimized by Polak-Ribiere algorithm to a RSM gradient of.05 kcal A−1 mol−1. For the 100 lowest energy conformationsound, an optimization of the geometry with a more stringent

inimization criterion (MM+, maximum energy gradient of× 10−4 kcal mol−1 A−1) was carried out. The 25 lowest energyonformations found were fully re-optimized in the Gaussian3 program package [5] by a DFT approach at the B3LYP level.he 6-31G* basis set was used for all the atom of molecules.

n order to confirm that the structures are in the local min-ma, we have calculated the normal mode frequencies of the

ptimized conformations. Each vibrational spectrum showso negative value of frequency, which suggests that the opti-ize structures are really in a minimum point of potential

nergy. The conformer populations were calculated based onhe Boltzmann equation.

7 ) 809–818 811

2.4. Chemistry

2.4.1. Preparation of the 3ˇ-acetoxy-9˛-methoxy-15˛-hydroxy-5˛-cholest-8(14)-en-7-one (4)i. According to Fieser [2]. To a stirred solution of 3�-acetoxy-

5�-cholesta-8,14-dien-7-one (1) (2.00 g, 4.53 mmol) in CHCl3(50 mL), m-chloroperoxybenzoic acid (mCPBA) (1.116 g of77% mCPBA, 4.98 mmol) in CHCl3 (30 mL) was added at 0 ◦C.The resulting mixture was stirred at 3 ◦C for 42 h. Then themixture was washed with a saturated aqueous solution ofNa2S2O3 (20 mL) and with a saturated solution of NaHCO3

(20 mL). The chloroform layer was dried over anhydrousNa2SO4 and the solvent evaporated at reduced pressure.The crude product obtained was dissolved in boiling anhy-drous MeOH (100 mL) and the solution was refluxed for5 min. The crystalline product formed after cooling, wasrecrystallized from MeOH to afford the compound (932 mg,42% yield) now identified as the 3�-acetoxy-9�-methoxy-15�-hydroxy-5�-cholest-8(14)-en-7-one (4), as colourlessprisms: m.p. 161–162 ◦C; [˛]D23 + 5.6 (CHCl3, c 1); UV (EtOH)�max 244 nm (ε 8600); IR 3340, 1723, 1662, and 1612 cm−1; [lit.[2] m.p. 160–161 ◦C; [˛]D23 + 4.3 (CHCl3, c 2.50)]; TLC [elutinghexane/AcOEt (80:20, v/v)]: Rf 0.35; EI m/z: 488 (6%; M+), 472(11 %), 471 (8 %), 457 (27 %); 456 (32 %), 441 (100 %), 376(3.5 %), 343 (15 %), 320 (42 %), 303 (44 %); ESI-MS (positive)m/z: 999 (100%, 2 × M + Na+), 511 (43%, M + Na+), 457 (28%,M + H+ − MeOH). Anal. calcd for C30H48O5: C, 73.73; H, 9.90.Found: C, 73.68, H, 9.85.

ii. By crystallization from methanol of pure 3ˇ-acetoxy-14˛,15˛-epoxy-5˛-cholest-8-en-7-one (2). The epoxy sterol 2 (600 mg,1.36 mmol) was refluxed in anhydrous methanol (30 mL)for 5 min. On cooling a crystalline compound (605 mg,91% yield) was obtained, which after recrystallizationfrom MeOH (585 mg, 88% yield), showed: m.p. 160–161 ◦C;[˛]D23 + 5.1 (CHCl3, c 1) and was identical in all aspects(mmp, TLC, 1H and 13C NMR) to the compound obtainedabove according to the Fieser’s procedure.

2.4.2. Acetylation of 3ˇ-acetoxy-9˛-methoxy-15˛-hydroxy-5˛-cholest-8(14)-en-7-one (4)Acetic anhydride (60 �L, 0.61 mmol) was added to a solu-tion of 15�-hydroxy sterol 4 (200 mg, 0.41 mmol) in pyridine(3 mL) containing 4-dimethylaminopyridine (50 mg). After 48 hat room temperature, the mixture was poured into ice coldhydrochloric acid (5 mL of 2 M HCl) and the steroid wasextracted with ethyl acetate (3 × 10 mL). The organic solu-tion was washed with water, dried over anhydrous Na2SO4

and the solvent was evaporated under reduced pressure. Theresidue was then purified by flash chromatography [elutingwith hexane/AcOEt (80:20, v/v)] to afford the 3�,15�-diacetoxy-9�-methoxy-5�-cholest-8(14)-en-7-one (8) (178 mg, 82% yield):an oil; [˛]D23 −34.0 (CHCl3, c 1); UV (EtOH) �max 241 nm(ε 8200); IR � 1723, 1668 and 1614 cm−1; TLC [eluting withhexane/AcOEt (80:20, v/v)]: Rf 0.22; ESI-MS (positive) m/z:1084 (100%, 2 × M + Na+), 553 (48%, M + Na+). Anal. calcd for

C32H50O6: C, 72.42; H, 9.50. Found: C, 72.50, H, 9.56.

Treatment of this compound (30 mg, 0.061 mmol) withmethanolic potassium hydroxide (1 mL; 0.1 M) for 48 h atroom temperature afforded a crude keto diol (25 mg, m/z 447),

( 2 0

812 s t e r o i d s 7 2

which by treatment with acetic anhydride in pyridine for24 h regenerates the 3�-acetoxy-9�-methoxy-15�-hydroxy-5�-cholest-8(14)-en-7-one (4).

2.4.3. Oxidation of 3ˇ-acetoxy-9˛-methoxy-15˛-hydroxy-5˛-cholest-8(14)-en-7-one (4)Pyridinium chlorochromate (PCC; 221 mg, 1.0 mmol) wasadded to a solution of the 15�-hydroxy sterol 4 (200 mg,0.41 mmol) dissolved in CH2Cl2 (5 mL). The mixture was stirredat room temperature for 24 h and then poured into an icecold solution of hydrochloric acid (5 mL of 0.5 M HCl) andextracted with ethyl acetate (3 × 10 mL). The organic solu-tion was dried over anhydrous Na2SO4 and the solvent wasevaporated under reduced pressure to afford a crude prod-uct which was purified by flash chromatography [elutingwith hexane/AcOEt (80:20, v/v)] to give the 3�-acetyloxy-9�-methoxy-5�-cholest-8(14)-en-7,15-dione (9) (155 mg, 78%yield): m.p. 152–153 ◦C; [˛]D23 + 102.9 (CHCl3, c 0.5); UV (EtOH)�max 250 nm (ε 5600); IR � 1721, 1696, 1644, 1600, 1473 cm−1;TLC [eluting with hexane/AcOEt (80:20, v/v)]: Rf 0.25; ESI-MS(positive) m/z: 996 (100%, 2 × M + Na+), 509 (43%, M + Na+). Anal.calcd for C30H46O5: C, 74.04; H, 9.53. Found: C, 74.18, H, 9.32.

3. Results and discussion

In the present study, we have reconsidered the reaction of3�-acetoxy-5�-cholesta-8,14-dien-7-one (1) with mCPBA, orig-inally described by Fieser et al. [2] and have isolated witha 42% yield, a compound whose physico-chemical prop-erties (m.p., [˛]D, UV, IR) were superimposable to thosedescribed by Fieser for its unidentified compound X, obtainedin the same reaction, after crystallization of the reac-tion mixture from methanol. This compound is differentfrom the 3�-acetoxy-5�-cholesta-14�,15�-epoxy-5�-cholest-8-en-7-one (2), previously isolated by us [1] (Scheme 1) aftercolumn chromatography purification of the reaction mixture(61% yield). The mass spectrum of compound X shows amolecular ion (m/z 488), which results from the addition of amolecule of methanol to the epoxide 2, the real product of thereaction. In fact, this hypothesis was simply demonstrated inour laboratory by dissolving the epoxide 2 in hot methanol andobserving the formation of a less soluble crystalline compoundwith the physico-chemical properties reported by Fieser et al.[2] for their unidentified compound. Moreover, the elementalanalysis of X was in perfect agreement with the molecular for-mula C30H48O5, thus supporting the hypothesis that elementsof methanol are likely added to the epoxide ring of compound2. In this work we unequivocally assign the structure of thereaction product of the methanol addition to the epoxiketone2, taking into consideration all four possible reaction products(Scheme 2).

In this process, UV and IR spectra analysis of compound Xsuggest the survival of the �8(9)-7-ketone system in the struc-ture. In fact, the UV spectrum showed a maximum (� 244 nm)indicative, on the base of the Woodward–Fieser’s rules [6–8],

of a �,�-unsaturated ketone in a �8(9)-7-ketone system (calcd[6–8] 249 nm), in good agreement whit the ones present instructures 6 and 7, in which the double bond is substitutedbut not exocyclic to any ring.

0 7 ) 809–818

A �8(14)-7-ketone system, as that present in the structures4 and 5 (in which the double bond is doubly exocyclic) shouldabsorb at higher values of wavelength (calcd [6–8] 259 nm).

Similarly, the IR evidences appeared to suggest the pres-ence of an �8(9)-7-ketone system (a transoid system), since the�C O band (at 1662 cm−1) was stronger than the �C C band (at1612 cm−1) and the separation of the two bands (60 cm−1) wasless than 75 cm−1 and therefore diagnostic of a transoid systemon the basis of some accepted evaluations [9]. On the con-trary, in the case of a �8(14)-7-ketone system (a cisoid system),the observed �C O and �C C bands should have been similar toeach other and their separation should have been larger than75 cm−1 [9].

On the other hand, these UV and IR behaviors have beenreported for some known steroidal �8(9)-7-ketones as the 5�-cholest-8(9)-en-7-one (�max 252 nm) [10], and its 3�-acetoxy[11] and 3�-hydroxy [12] derivatives (�max 253 nm for both),as the 3�,11�,15�-triacetoxycholest-8-en-7-one (�max 249 nm)[13] and the steroid 2 (�max 246 nm) [1], the parent of the Fiesercompound X.

Moreover, the information that could be derived fromroutinary 1H and 13C NMR spectra of compound X was onlyindicative of its origin but unsuitable for a clear-cut identi-fication of its structure. In addition, the 1H NMR spectrumof the compound X exhibits several relevant signals, in addi-tion to the acetoxylic methyl group signal, a singlet (3.13 ppm)assigned to the protons of a methoxy group, a signal (4.72 ppm,dddd) assigned to the 3�-proton and a broad singlet (5.16 ppm)exchangeable with D2O. This last signal, in the resolution-enhanced spectrum, appears as a double doublet (J = 1.8 and1.8 Hz) attributable to an OH proton. The spectrum also showsa double doublet at 4.48 ppm (J = 6.5 and 1.8 Hz) which col-lapsed to a doublet (J = 6.5 Hz), either by irradiation of the OHproton signal, or by addition of D2O. This could be attributedeither to a proton geminal to a hydroxyl group or to a protonlocated on a carbon adjacent to another bearing a methoxygroup. Finally, the 1H NMR spectrum showed the presence ofa complex signal (at 2.70 ppm, dddd), which could be assignedto the 5�-proton of the steroid. The 13C NMR spectrum wasin good agreement with the presence of an acetoxy group(signal at 170.4 ppm) and of an �,�-unsaturated ketone (sig-nals at 206.1, 168.7 and 131.3 ppm). At this point, consideringthat Fieser et al. [2] were unsuccessful in the acetylationof the compound X, under common conditions (treatmentwith Ac2O in pyridine for 48 h at 25 ◦C), and that C. Djerassiand E.J. Taylor reported [13] that the 15�-hydroxy group ofa steroidal �8(9)-7-ketone (3�,11�,15�-trihydroxy-5�-cholest-8(9)-en-7-one) is indeed easily acetylated under similarconditions, it seemed more plausible to attribute structure 6 tocompound X.

In effect, in our hands, compound X could be acetylatedonly in the presence of dimethylaminopyridine (DMAP), a baseknow to allow the acetylation of tertiary steroidal alcohols [14],thus supporting the assignment of structure 6 to the unknowncompound.

The obtained diacetate Y (Scheme 3) was saponified with

methanolic potassium hydroxide to afford a keto diol which,by acetylation with acetic anhydride in pyridine, at room tem-perature overnight, afforded the compound X reported byFieser.

s t e r o i d s 7 2 ( 2 0 0 7 ) 809–818 813

Scheme 3 – Reactivity of the compound X and structures oft

att

urlasw7stsokmmtt

cbbsofuX

p(

32

C(so1

et

Table 1 – 1H NMR chemical shifts (ppm) assignment forcompounds 4, 8 and 9

Proton 4 4 8 9CDCl3 C6D6 CDCl3 CDCl3

1� 1.90 1.79 1.89 1.921� 1.43 1.15 1.47 1.512� 1.92 1.90 1.90 1.912� 1.55 1.40 1.50 1.483� 4.72 4.77 4.71 4.734� 1.73 1.50 1.70 1.764� 1.35 1.13 1.33 1.435� 2.70 2.51 2.66 2.626� 2.30 2.08 2.21 2.346� 2.09 1.71 1.98 2.3911� 1.74 1.40 1.77 1.7611� 1.97 1.62 2.08 1.9612� 1.67 1.54 1.69 1.5812� 2.02 1.76 2.04 2.1315� 4.48 4.57 5.68 –16� 1.99 2.25 1.90 2.5916� 1.66 1.52 1.73 2.0417� 1.83 1.99 1.67 1.7120 1.47 0.65 1.48 1.6222a 1.47 0.62 1.36 1.3422b 1.10 1.43 1.08 1.1223a 1.40 0.95 1.33 1.3323b 1.18 1.46 1.16 1.1924 H2 1.13 1.13 1.11 1.1425 1.51 1.46 1.51 1.5118-Me 0.868 1.227 0.886 0.96819-Me 1.015 1.173 0.994 1.00821-Me 0.951 1.510 0.953 1.00226-Me 0.857a 0.913a 0.853a 0.870a

27-Me 0.861a 0.913a 0.848a 0.865a

CH3COO 2.02 1.76 2.01 2.02OCH3 3.13 3.03 3.15 3.18

he formed compounds 8 and 9.

At this point, we thought to exclude the oxidability of thelcoholic group of the compound X, under controlled condi-ions, in order to unequivocally reject structure 7 in favor tohe isomeric structure 6.

Surprisingly, compound X could be oxidized (Scheme 3),nder mild conditions (pyridinium chlorochromate, CH2Cl2,oom temperature), to afford a diketone Z having the molecu-ar formula C30H46O6 (mass spectrum and elemental analysis)nd containing the methoxy group (by NMR evidence). Thisuggested that the hydroxyl group in the starting molecule Xas a hindered secondary alcohol such as that of compound. Moreover, the UV spectrum of the obtained ketone showed aignificant shift of the absorption maximum when comparedo the parent compound X (from 244 nm to 250 nm). This UVhift could be explained by the assumption that the oxidationf the hydroxy group of compound 7 to the correspondingetone was associated with conformational changes of theolecule, which resulted in a greater coplanarity of the chro-ophore. Consequently, the UV maximum would approach

he calculated [6–8] value (249 nm) for the �8(9)-7-ketone sys-em present in compound 7.

Alternatively, the shift in the UV absorption maximuman be simply explained by an extension of the chromophorey the formed carbonyl group. This hypothesis is supportedy the observation that steroidal �8(14)-7,15-diketone systemshow a UV maximum at 255 nm [15]. In this case, despite previ-us considerations, structures 4 and 5 should be reconsideredor compound X. This justifies the difficulties experienced bys [1] and others [2] to assign the correct structure to steroid, using traditional physicochemical methodology.

Thus, we decided to deeply study the structure of com-ound X using two-dimensional nuclear magnetic resonance

2D NMR) supported by a molecular modeling study.

.1. 1H and 13C signal and structural assignment byD NMR spectra

omplete proton and carbon resonance assignmentsTables 1 and 2) for compounds X, Y and Z (of assignedtructures 4, 8 and 9) were achieved using a combination

f 1D and 2D NMR experiments (COSY, TOXY, HSQC, HMBC,H–1H J-resolved and NOESY). For compound X (4), all NMRxperiments were performed for both CDCl3 and C6D6 solu-ions. In fact, we observed that the 1D proton spectrum for

OH 5.16 5.46 – –

a These assignments may be interchanged.

the C6D6 solution exhibits a higher number (10) of isolatedhydrogen signals with respect to those present in the spec-trum recorded for the CDCl3 solutions (6). However, in thespectrum recorded for C6D6 solution, the resonances of theremaining hydrogen atoms show an overlapping in a smallerresonance range, making their assignments more difficult.

The chemical shifts of all protons were derived fromresolution-enhance 1D spectra or from F2 projections in1H–1H J-resolved 2D spectra. Using a combination of 2Dshift-correlated experiments (COSY and TOCSY for 1H–1Hcorrelation and HSQC for 1H–13C correlation), and startingfrom the characteristic 3�-H signal, we were also able toassign the resonance of all protons and carbons of the C-1–C-6 steroidal fragment. Similarly, starting from the hydroxylicproton signal and from those of the protons of the 21, 26and 27 methyl groups, we assigned the resonance of all pro-tons and carbons of the C-15–C-16–C-17–C-20 fragment andthose of the steroidal side chain. The analysis of the HMBCspectra allowed the completion of the lacking connectivities,

since the cross-peaks revealed two- or three-bond correlationsbetween protons and carbons (Table 3). More specifically, start-ing from three-bond correlation of 5�-proton we were able toidentify the signal of C-19 carbon and those of the bonded

814 s t e r o i d s 7 2 ( 2 0

Table 2 – 13C NMR chemical shifts (ppm) assignment forcompounds 4, 8 and 9

Carbon 4 4 8 9CDCl3 C6D6 CDCl3 CDCl3

1 28.4 28.7 28.5 26.72 27.0 27.5 27.0 28.53 72.5 72.6 72.6 72.44 33.3 33.6 33.3 33.65 33.6 34.0 33.2 36.36 44.7 44.8 44.8 46.17 206.1 205.4 199.7 204.48 131.3 131.5 132.4 141.89 78.2 78.4 78.4 80.810 40.9 41.1 40.8 41.811 25.0 25.4 25.4 25.012 34.1 34.8 34.3 34.913 44.6 44.8 44.2 43.514 168.7 168.7 161.7 149.315 69.9 70.1 72.9 202.716 36.2 36.9 36.2 41.017 53.7 54.1 53.6 51.518 18.2 18.3 18.8 16.519 15.3 15.2 15.4 15.120 33.4 34.0 33.3 34.721 18.9 19.2 18.9 18.922 35.9 36.4 35.9 35.623 23.9 24.3 23.6 23.424 39.3 39.8 39.3 39.325 27.9 28.3 27.9 28.026 22.5a 22.7a 22.5a 22.5a

27 22.8a 23.0a 22.7a 22.7a

CH3COO 170.4 169.4 170.5 170.4CH3COO – – 170.2 –CH3COO 21.3 21.0 21.3 21.3CH3COO – – 21.2 –OCH3 51.5 51.4 51.6 52.7

a These assignments may be interchanged.

Table 3 – HMBC and NOESY responses of compound 4

Proton HMBC

1� C-2; C-3; C-9; C-10; C-191�

2� C-1; C-102�

3� C-1; C-2; C-4; C-5; MeCOO4�

4�

5� C-1; C-3; C-4; C-6; C-7; C-9; C-10; C-196� C-4; C-5; C-7; C-8; C-106� C-4; C-8; C-1011� C-9; C-10; C-1311� C-9; C-1312� C-11; C-13; C-1812�

15� C-8; C-13; C-16; C-1716� C-8; C-13; C-14; C-15; C-1716�

17� C-12; C-13; C-16; C-18; C-20; C-21; C-2218-Me C-12; C-13; C-14; C-1719-Me C-1; C-5; C-9; C-1021-Me C-17; C-20; C-22OCH3 C-9OH C-14; C-15; C-16

0 7 ) 809–818

protons (by HSQC spectra). Consequently, the only other signalattributable to methylic protons was assigned to the 18-methylgroup.

The position of the methoxy group was determined con-sidering that, apart from the stereochemistry, the signalattributable to its protons showed a three-bond correlationwith a quaternary carbon bearing it. The position 9 for thiscarbon was derived considering that it shows, inter alia, three-bond connections with both the protons at C-19 and at C-5�.

At this point, we decided that only structures 4 and5 should be considered for compound X. Accordingly, theprotons at C-18 and at 16�-positions showed three-bond con-nections with the �-carbon of the �,�-unsaturated ketonesystem, thus indicating that this carbon should occupy posi-tion 14 in the steroid nucleus.

The position 8 for the �-carbon of the �,�-unsaturatedketone system was then derived from two three-bond correla-tions of this carbon with the 6�- and the 15�-protons. Finally,the position for the carbonylic carbon was suggested by twointense connections with the 5�-protons (three bonds) and the6�-protons (two-bond).

The assignment of the C-11 and C-12 resonances togetherwith those of the bonded protons completes the unequivocalassignment of the constitutional structure to the steroid.

Moreover, HMBC experiments did not allow us to assignthe stereochemistry of the methoxy group and to discriminatebetween the two epimeric structures 4 and 5, both containinga �8(14)-7-keto system.

Instead, NOESY experiments allowed us to unequivo-cally attribute structure 4 to Fieser’s compound. In fact,the NOE contacts observed in the NOESY spectra (Table 3),

corresponding to distances of <3 A (as measured on the com-puted molecule model of structure 4) showed cross-peaksbetween the methoxy protons and the protons at 5�-, 11�-,12�- and 17�-positions. The �-geometry of all, which was

NOESY

H-3�; H-5�

H-11�

Me-19H-1�; H-5�

H-6�

H-6�; Me-19H-1�; H-3�; OCH3

H-4�

H-4�; Me-19H-1�; OCH3

Me-18; Me-19OCH3

Me-18; Me-21Me-18; Me-21H-22a; H-22bH-22b; Me-18H-22b; Me-21; OCH3; OHH-11�; H-12�; H-15�; H-16�; H-20; Me-21H-2�; H-4�; H-6�; H-11�

H-12�; H-15�; H-!7�; Me-18H-5�; H-11�; H-12�; H-17�; OHH-17�; OCH3

s t e r o i d s 7 2 ( 2 0 0 7 ) 809–818 815

Table 4 – 1H–1H NMR coupling constants (J in Hz) for compounds 4, 8 and 9

Coupled protons 4 4 8 9Experimental Calculateda Experimental Experimental

1�,1� 13.0 13.2 ndb

1�,2� 4.1 3.9 4.1 ndb

1�,2� 13.4 13.2 13.8 ndb

1�,2� 2.5 2.7 2.8 ndb

1�,2� 3.9 3.5 3.9 ndb

2�,2� 14.3 13.9 ndb

2�,3� 4.9 4.8 5.1 4.92�,3� 11.3 10.8 11.3 11.63�,4� 4.9 4.8 4.9 4.93�,4� 11.1 10.8 11.3 11.64�,4� 13.2 13.0 13.54�,5� 3.5 2.9 3.6 3.54�,5� 12.8 12.6 13.0 12.65�,6� 4.0 3.9 4.2 4.85�,6� 12.8 12.5 12.8 12.66�,6� 16.4 15.8 15.011�,11� 14.5 14.5 ndb

11�,12� 4.7 4.3 5.0 ndb

11�,12� 2.5 2.4 3.3 2.511�,12� 13.0 12.9 13.4 ndb

11�,12� 4.6 4.5 4.9 4.912�,12� 13.0 13.2 13.315�, OH 1.8 – –15�,16� <1 0.7 <1 –15�,16� 6.5 6.6 6.5 –16�,16� 13.3 13.5 19.516�,17� 5.5 5.1 5.3 8.016�,17� 12.7 12.2 12.9 11.616�,OH 1.8 – –

e in i

e the

diapp(c�

aac

tpctFato

Uaa33

a Values calculated with the Altona equation [22] from dihedral angllevel).

b nd indicate that the coupling constant was not determined becaus

erived from some cross-peaks, indicates a spatial proxim-ty between the 5�-proton and the protons at positions 1�

nd 3�, between the 19-methyl protons and the protons atositions 2�, 4�, 6� and 11� and between the 18-methylrotons and the protons at positions 11�, 12�, 15�, 16�

Table 3). These interaction clearly suggested an � stereo-hemistry for the methoxy group and also confirmed theand � stereochemistry of the geminal methylene protons

nd the � stereochemistry of the 15 hydroxyl group. Thisllowed us to unequivocally assign structure 4 to the Fieserompound.

All vicinal coupling constants observed for the protons ofhe steroidal nucleus of 4 are in agreement with the valuesredicted (Table 4) using the results of the computer assistedonformational analysis performed on structure 4 (see Sec-ion 3.2). This definitively allowed us to assign structure 4 toieser compound X. Similarly, complete 1H and 13C resonancessignments (Tables 1 and 2) allowed us to associate struc-ures 8 and 9 to the compounds obtained by acetylation andxidation of the ketol 4.

Theoretical calculations were also useful to rationalize theV absorption values observed for compounds 4 and 8, which

re noticeably lower than both the calculated values [6–8]nd those observed for similar steroidal structures as the�-acetoxy-5�-cholest-8(14)-en-7-one (10; Fig. 1) [16] and the�,15�-diacetoxy-5�-cholest-8(14)-en-7-one (11) [17]. In fact,

solate global minimum (theoretical calculations at the B3LYP/6-31G*

signal was obscured by overlapping.

the inspection of the values of the torsional angle of the �,�-unsaturated system of compounds 4, 8, 10 and 11, obtainedfrom the conformational searches and geometry optimiza-tions at the B3LYP/6-31G* level, showed that a significantdeviation from the coplanarity of the carbonyl and the con-jugated double bond is present in compounds 4 and 8. On thecontrary, these groups are nearly coplanar in compounds 10and 11. This geometry causes a reduction of the conjugationand is consequently responsible for the ipsochromic shift ofthe �max value observed for compounds 4 and 8. On the otherhand, the presence of a strong hydrogen bond between the car-bonyl oxygen and the alcoholic hydrogen of 4 slightly reducesthe ipsochromic effect in this compound as compared to itsacetate 8.

Considering the high shift of �max observed between com-pound 8 and 11, it also appears possible to attribute theobserved distortion of planarity of the �,�-unsaturated systemin 4 and 8 to the presence of the methoxy group at position9�.

As a final observation we propose that the mechanismdepicted in Scheme 4 may properly illustrate the formationof compound 4. Clearly, the possible concurrent attack of

methanol from the �-side of the molecule does not occur dueto the steric hindrance of 18- and 19-methyl groups. More-over, the absence of a possible 14� attack could be rationalizedconsidering the hindrance resulting from the hydrogen bond

816 s t e r o i d s 7 2 ( 2 0 0 7 ) 809–818

Scheme 4 – Possible mechanism

Fig. 1 – Structure, torsional angle of the chromophore, andwas derived by Altona and coworkers [22]. The calculatedvalues for these constants are in agreement with those exper-imentally observed, thus strongly supporting the assignmentof structure 4 to Fieser’s X compound.

UV absorption maximum of compounds 4, 8, 10, and 11.

between the ketonic oxygen and the 15�-hydroxy groupobserved by theoretical calculation.

This mechanism is in good agreement with the behaviorof other steroidal 14�,15�-epoxides we encountered in someprevious studies [1,17–19].

3.2. Theoretical calculations an conformational

analysis of structure 4

A conformational analysis of structure 4 identified severalconformations with energy close to that of the global min-

of formation of compound 4.

imum (Table 5). For each conformation we determined therelative energy, the percentage contribution to the overall pop-ulation through the Boltzmann equation and the values of themore significant torsional angles.

We found that the A,B trans junction and the presence ofdouble bond at 8(14) position makes structure 4 particularlyrigid. In fact, all the selected conformers showed superimpos-able shapes for the steroidal nucleus, while they differ onlyfor the torsional angles of the 3�-acetoxy group and of theside chain (Fig. 2). All conformers showed an intramolecu-lar hydrogen bond involving the proton of the hydroxy groupat C-15 and the keto group at C-7 with the following geome-try for the C O· · ·H O system: O· · ·H distance = 1.845 A; H Odistance = 0.979 A; O· · ·H O angle = 146◦.

Ring conformations were determined using theCremer–Pople puckering parameters [20] which definethe conformation of a puckered ring in a quantitative andmathematically well defined way [21]. There are three puck-ering parameters for a six member ring (Q—the amplitudeof puckering, and two phase angles, � and ϕ), while for thefive-member ring there is just one amplitude-phase pair (q2

and ϕ2).The puckering parameters indicate for ring A (Q = 0.57; � = 2;

ϕ = 256) a 1�,4�-chair conformation, for the ring B (Q = 0.54;� = 161; ϕ = 250) an intermediate structure between 5�,8�-chairand 10�-sofa, for the ring C (Q = 0.45; � = 48; ϕ = 232) a 12�-sofaconformation and for the ring D (q2 = 0.39; ϕ2 = 138) a 17�-envelope conformation.

Considering that the equilibrium conformers differ fromeach other only for the conformations of the acetoxy groupand of the side chain, the vicinal coupling constants ofthe nucleus protons (Table 4) were calculated from the cor-responding H C C H dihedral angles determined in theconformation 4A, using a Karplus analogues equation which

Fig. 2 – Steroidal backbone (non-H atoms) of the lowestenergy conformation (4A) of compound 4. The other morepopulated conformations differ only by the shown torsionalangles values (Table 5).

s t e r o i d s 7 2 ( 2 0 0 7 ) 809–818 817

Table 5 – Relative energies (Erel), equilibrium percentages (%), and selected torsional angles (�) of the lowest energyconformations of compound 4

Conformer Erel (kcal/mol) %a Torsional anglesb (◦)

�1 �2 �3 �4 �5 �6 �7 �8 �9 �10

4A 0.000 9.4 −55 39 −51 −26 58 −171 174 176 −174 854B 0.063 8.5 −55 38 −53 −28 57 −170 174 −176 −65 844C 0.077 8.3 −55 38 −52 −27 54 60 174 −177 −63 1544D 0.090 8.1 −55 38 −53 −27 56 −171 175 −176 −65 1544E 0.091 8.1 −55 38 −52 −27 54 60 178 177 −170 854F 0.106 7.9 −55 38 −52 −27 54 60 175 −177 −63 854G 0.118 7.7 −56 39 −51 −26 58 −172 174 175 −174 1534H 0.148 7.3 −55 38 −52 −27 54 63 180 177 −173 1544I 0.621 3.3 −55 38 −52 −27 56 −177 168 60 −177 844L 0.717 2.8 −55 39 −51 −27 57 −178 59 179 −65 1544M 0.729 2.8 −55 39 −51 −27 53 62 −177 −63 −59 844N 0.760 2.6 −55 38 −52 −27 56 −176 168 59 −177 1544O 0.788 2.5 −56 39 −52 −28 54 60 172 63 −175 1544P 0.809 2.4 −55 38 −51 −28 56 −177 60 171 −173 1544Q 0.829 2.3 −56 38 −51 −27 55 −178 62 179 −65 854R 0.907 2.0 −55 38 −52 −26 56 −171 178 −64 −59 854S 0.918 2.0 −55 39 −52 −28 54 60 170 63 −175 854T 0.925 2.0 −56 38 −51 −27 54 −179 61 170 −174 85

tionC-9–CC-24–

A

Tttaf

r

a Percentage contribution to the overall population of each conformab Torsional angles: �1 = C-10–C-1–C-2–C-3; �2 = C-5–C-6–C-7–C-8; �3 =

�6 = C-17–C-20–C-22–C-23; �7 = C-20–C-22–C-23–C-24; �8 = C-22–C-23–

cknowledgments

his work was financially supported by the Italian Minis-ero dell’Universita e della Ricerca. This work is dedicated tohe memory of the Professors Louis and Mary Fieser who, inbsence of clear evidence, never proposed tentative structuresor steroids.

e f e r e n c e s

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determined through the Boltzmann equation.-11–C-12–C-13; �4 = C-14–C-15–C-16–C-17; �5 = C-16–C-17–C-20–C-22;C-25; �9 = C-23–C-24–C-25–C-26; �10 = CO–O–C-3–C-2.

[10] Midgley I, Djerassi C. Mass spectrometry in structural andstereochemical problems. Part CCXX. Synthesis and massspectra of 5a-cholest-8(9)- and 8(14)-en-7-ones. J Chem SocPerkin 1972;1:2771–6.

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[12] Tsuda M, Parish EJ, Schroepfer GJ. Carbon 13 nuclearmagnetic resonance studies of allylic hydroxysterols.Assignement of structure to5�-cholest-8(14)-ene-3�,7�,15�-triol, an inhibitor of sterolsynthesis. J Org Chem 1979;44:1282–9.

[13] Taylor J, Djerassi C. Synthesis ofcholest-5-ene-3�,11�,15�-triol-7-one. A model for steroidnucleus of oogoniols, a sex hormone of water mold Achlya. JOrg Chem 1977;42:3571–9.

[14] Hofle G, Steglich W, Vorbruggen H. 4-Dialkylaminopyridinesas higly active acylation catalysts. Angew Chem Int Ed Engl1978;17:569–83.

[15] Dorfman L. Ultraviolet absorption of steroids. Chem Rev1953;53:47–144.

[16] Wintersteiner O, Moore M. The dehydration of the7-epimeric 3(�)-acetoxycholestanols-7. Some transformationproducts of �-cholestenol. J Am Chem Soc 1943;65:1507–13.

[17] Anastasia M, Cighetti G, Manzocchi Soave A. Reactionsof steroidal 8�,9�- and 8�,14�-epoxy-7-ketones withacetic acid. J Chem Soc Perkin 1977;1:427–9.

[18] Anastasia M, Allevi P, Fiecchi A, Scala A. 15-Oxygenatedsterols by m-chloroperbenzoic acid oxidation of3�-acetoxy-5�-cholesta-8,14-diene. J Org Chem1981;46:3265–7.

[19] Anastasia M, Allevi P, Fiecchi A, Galli G, Gariboldi P, Scala A.

Synthesis of 11- and 15-oxygenated steroids. The course of8,14-dienes oxidation by chromic acid. J Org Chem1983;48:686–9.

[20] Cremer D, Pople JA. A general definition of ring puckeringcoordinates. J Am Chem Soc 1975;97:1354–8.

( 2 0

818 s t e r o i d s 7 2

[21] Cremer D. On the correct usage of the Cremer–Poplepuckering parameters as quantitative descriptors of ringshapes—a replay to recent criticism by Petit, Dillen andGeise. Acta Cryst 1984;B40:498–500.

0 7 ) 809–818

[22] Haasnoot CAG, De Leeuw FAAM, Altona C. The relationshipbetween proton–proton NMR coupling constants andsubstituent electronegativities-I; an empirical generalizationof the Karplus equation. Tetrahedron 1980;36:2783–92.