Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 56–62

A comparative molecular modeling study of dydrogesterone with otherprogestational agents through theoretical calculations and nuclear

magnetic resonance spectroscopy

Diego Colomboa, Patrizia Ferraboschia,∗, Paolo Prestileoa, Lucio Tomab

a Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universita di Milano, Via Saldini 50, 20133 Milano, Italyb Dipartimento di Chimica Organica, Universita di Pavia, Via Taramelli 10, 27100 Pavia, Italy

Received 25 February 2005; accepted 12 July 2005

Abstract

6-Dehydroretroprogesterone (dydrogesterone) and three other natural or synthetic progestins (progesterone, retroprogesterone, and 6-dehydroprogesterone) were submitted to a conformational study through theoretical calculations at the B3LYP/6-31G* level and high fieldNMR spectroscopy. The study allows to define the role of the two structural features which differentiate these steroids, i.e., the C9 and C10c nfigurationa lectivity ofd©

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onfiguration and the C6–C7 unsaturation. The combined effects of the conformational preference of A ring, determined by the cot C9 and C10, and the enhanced rigidity due to the C6–C7 double bond, could account both for the higher activity and seydrogesterone with respect to the other three steroids.2005 Elsevier Ltd. All rights reserved.

eywords: Dydrogesterone; Progestins; Retrosteroids; Molecular modeling; DFT calculations; NMR

. Introduction

Beside progesterone1, the natural progestational agentainly produced bycorpus luteum, many synthetic pro-estins are known, classified into different groups on theasis of their chemical structure and biological effect[1,2].he early recognition that the 10�-methyl group of1 is actu-lly detrimental to the activity has led to steroids structurallyodified in this group but still endowed with progestationalffects: in some cases, the methyl group is substituted byhydrogen atom, yielding the 19-norsteroids class; another

lass is represented by retrosteroids, e.g., retroprogesterone, characterized by an inversion of configuration at C9 and10[3,4]. Compound2, in spite of the “unnatural” skeletonearing the 10-methyl group on the� side of the molecule,etains the biological activity, i.e., the progestational effect,imilarly to other retrosteroids, pharmacologically active asnabolic, antiandrogenic, and salt-retaining agents[4,5].

∗ Corresponding author. Tel.: +39 02 50316045; fax: +39 02 50316040.E-mail address: patrizia.ferraboschi@unimi.it (P. Ferraboschi).

The most representative compound among the reteroids with progestational activity is certainly 6-dehydrotroprogesterone (dydrogesterone)3, which possessesbiological potency significantly higher than progesteritself. Since it binds almost exclusively to the progestereceptor, it can activate only the effects depending onone. Moreover, as a consequence of its better bioavailaand the progestational activity of its main metabol(20-, 21-, and 16-hydroxy derivatives), its equivalent dregarding the endometrial proliferation, is 10–20 times lothan that of progesterone itself[2].

The inverted configuration at C9 and C10 andadditional C6–C7 double bond are the two structural feawhich differentiate dydrogesterone3 from progesterone1;a comparison of these two compounds with those beonly one of the two features might allow to determine tinfluence on the activity; retroprogesterone2, already mentioned, and 6-dehydroprogesterone4 are known compoundThe latter is surprisingly less active than progesterone1 [6],indicating that unsaturation alone is not responsible foactivity enhancement of3; compound2, instead, is know

960-0760/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.oi:10.1016/j.jsbmb.2005.07.009

D. Colombo et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 56–62 57

to have progestational activity higher than1 though lowerthan3 [3].

As regards to the effects of the inversion of configura-tion at C9 and C10 on the geometry of these steroids, abent shape has been suggested for3, contrarily to1 whichhas an almost planar geometry[2]. However, thecis fusionbetween B and C rings in2 and3 makes this bicyclic por-tion of the tetracyclic skeleton acis-decaline-like fragmentw les;i ighte ailedm -l nt ecentc al-c relr om-p DFTl ereu

2

2

ro-gg6r gt

2

n03[ om-

pounds1–4 was explored through optimization of all thepossible starting geometries which were optimized withinthe DFT approach at the B3LYP level with the 6-31G*

basis set. All the degrees of conformational freedom wereconsidered, in particular the possible existence of differ-ent chair or twisted-boat conformations at the A, B, andC hexacyclic rings as well as the orientation of the acetylside chain at C17. Several conformations were locatedfor each compound;Table 1 summarize the main geo-metrical data together with the relative energy and thepercentage contribution to the overall population of eachconformation determined through the Boltzmann equa-tion. The geometry of A–C rings is described throughtwo kind of descriptors, i.e., a significant torsion anglefor each ring,τA, τB, τC, and the ring puckering coor-dinates determined according to Cremer and Pople[12],also determined for D ring. The orientation of the C17side chain is described through the torsional angleτS(C16–C17–C20–O20). The most populated conformationsof each compound are also represented as three-dimensionalplots inFig. 1.

2.3. NMR spectroscopy

All NMR spectra were recorded at 298 K with a BrukerA and1 mb n theδ ra ).C( bya SYe Thep1 tedf ppm( t)a -l erea ):r -m ne TheN .0 sa cantd es( Ds eryd Kz nt,s aina ain.A arep

hich could enhance the flexibility of the entire molecun principle, more than one accessible conformation mxist for these two compounds. To our knowledge, no detodeling study on3, as well as on2 and4, has been pub

ished while the conformational properties of1 have beehoroughly discussed along the years though the most romplete study is limited to the semiempirical level of culations[7] and ab initio or density functional results aimited to the global minimum conformation[8]. Here, weeport a comparative conformational study of the four counds performed through theoretical calculations at the

evel; in addition, high field NMR experimental data wsed to support the results of the calculations.

. Material and methods

.1. Materials

Progesterone1 was purchased from Sigma. Retropesterone2 was prepared starting from 11�-hydroxypro-esterone (Sigma) according to a reported method[5,9]. The-dehydro compounds3 and4 were obtained from2 and1,espectively, by reaction with chloranil int-butanol accordino published procedures[10].

.2. Theoretical calculations

All calculations were carried out using the Gaussia11] program package. The conformational space of c

VANCE-500 spectrometer operating at 500.1325.76 MHz for 1H and 13C, respectively, using a 5 mroadband reverse probe. Chemical shifts are reported o(ppm) scale and are relative to pyridine signals (7.19 fo1Hnd 123.50 ppm, central line, for13C spectra, respectivelyompounds1–4 (ca. 12 mg) were dissolved in pyridine-d5

0.5 mL) under N2, and their assignments were givencombination of 1D and 2D COSY, HMQC and NOE

xperiments, using standard Bruker pulse programs.ulse widths were 9.6�s (90◦) and 8.4�s (90◦) for 1H and3C, respectively. Typically 32 K data points were collecor one-dimensional spectra. Spectral widths were 11.455733 Hz) for1H NMR (digital resolution: 0.17 Hz per poinnd 259.84 ppm (32,680 Hz) for13C NMR (digital reso

ution: 0.1 Hz per point). 2D experiments parameters ws follows. For1H–1H correlations (COSY and NOESYelaxation delay 2.0 s, data matrix 1 K× 1 K (512 experients to 1 K zero filling in F1, 1 K in F2), eight transients iach experiment, spectral width 6.4 ppm (3205.13 Hz).OESY spectra were generated with a mixing time of 1nd acquired in the TPPI mode. There were not signifiifferences in the results obtained at different mixing tim0.5–1.5 s). For13C–1H correlations (HMQC, using BIRequence)[13]: relaxation delay 2.0 s, inversion recovelay 0.5 s, data matrix 1 K× 1 K (256 experiments to 1ero filling in F1, 1 K in F2), 32 transients in each experimepectral width 6.4 ppm (3205.13 Hz) in the proton domnd 154.09 ppm (19379.57 Hz) in the carbon domll 2D spectra were processed with the Bruker softwackage.

58 D. Colombo et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 56–62

Fig. 1. Three-dimensional plot of the most significant conformations of compounds1–4.

3. Results

3.1. Conformational properties of progesterone 1

The global minimum of progesterone1, located in thepresent study, corresponds, in the tetracyclic skeleton, to thegeometry already determined at the same level of calcula-tions [8]. A careful analysis of the conformational freedomof the various rings allowed to verify that thetrans fusion atthe ring junctions makes quite rigid the overall structure, inparticular rings B, C, and D. In fact, B ring inversion from the5�,8�-chair to a twisted-boat conformation (e.g.,1A → 1E)is disfavored by more than 6 kcal/mol (Table 1) and evenmore difficult (11 kcal/mol) is the same inversion of C ring(e.g.,1A → 1G). The global minimum1A shows a 1�,2�-half-chair geometry at A ring; its inversion (e.g.,1A → 1C)is quite easier than that of B and C rings as it costs only about2 kcal/mol; thus, conformations1C and1D, presenting the a1�,2�-half-chair geometry at A ring, give a small contribu-tion (about 4%) to the overall population. The energy barrierfor A ring inversion was also determined by locating the tran-sition state of the conformational interconversion1A → 1Cand its height was 7.04 kcal/mol at the present level of calcu-lations, a value somewhat higher than the barrier determinedat the AM1 level (4.6 kcal/mol)[7]. The C17 side chain can

assume two energy minimum orientations, the preferred one,in agreement with literature X-ray, CD, and computationaldata[14–16], with a torsional angleτS of −32◦, the secondone, withτS of −138◦, about 1 kcal/mol higher in energy (see1A versus1B or 1C versus1D).

3.2. Conformational properties of retroprogesterone 2

The inversion of configuration at C9 and C10 has a relevantinfluence on the conformational properties of the molecule.In fact, A ring shows in the most populated conformations2Aand2B the 1�,2�-half-chair geometry, opposite to that foundin 1. Also in this case, A ring inversion is possible at a cost(2A → 2C, 2.35 kcal/mol) comparable to that found in1 andwith a similar energy barrier (6.64 kcal/mol). The tetracyclicskeleton, in particular the B/C ring portion, becomes moreflexible than in1. The inversion of the chair conformationof B ring of 2 becomes much easier as it can be seen fromTable 1. In fact, this transformation costs only 2.64 kcal/mol(2A → 2E); also, the contemporaneous inversion of B andC rings requires 3.54 kcal/mol (2A → 2I) and that of all thethree hexacyclic rings only 3.21 kcal/mol (2A → 2G). Thisallows a small contribution to the overall population of allthe conformations deriving from the inversion of the geom-etry of one or more rings though also in this case, as in

D. Colombo et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 56–62 59

Table 1Selected geometrical features, relative energies, and equilibrium percentages of the minimum energy conformations of compounds1–4

Erel (kcal/mol) % τS (◦) τA (◦) τB (◦) τC (◦) Ring puckering coordinates

A ring B ring C ring D ring

Q φ2 θ Q φ2 θ Q φ2 θ q2 φ2

1A 0.00 80.25 −32 −54 53 −54 0.44 17 54 0.54 169 6 0.57 267 5 0.47 1901B 0.97 15.64 −138 −54 53 −53 0.44 17 54 0.54 170 6 0.57 269 6 0.47 1861C 1.86 3.46 −32 55 54 −55 0.44 203 125 0.58 348 5 0.58 291 4 0.47 1911D 2.86 0.65 −138 55 55 −54 0.44 203 125 0.58 349 5 0.58 294 4 0.47 1861E 6.25 0.00 −31 −48 −51 −55 0.45 348 53 0.72 261 84 0.59 336 5 0.48 1911F 7.12 0.00 −136 −48 −52 −54 0.45 347 54 0.72 261 84 0.59 336 5 0.48 1861G 11.48 0.00 −34 −54 53 46 0.44 12 54 0.56 183 12 0.74 325 79 0.50 1951H 12.28 0.00 −136 −54 53 46 0.44 12 54 0.56 182 13 0.74 324 79 0.50 1921J 14.31 0.00 −32 55 56 46 0.44 208 125 0.60 252 1 0.74 328 78 0.50 1961K 15.03 0.00 −137 56 56 45 0.44 207 125 0.60 240 1 0.74 327 78 0.50 1921L 20.29 0.00 −32 −45 −37 46 0.47 336 56 0.68 246 79 0.75 328 78 0.50 1961M 21.09 0.00 −137 −45 −38 45 0.47 337 56 0.68 247 79 0.74 328 78 0.50 192

2A 0.00 79.61 −31 53 −59 −50 0.44 189 126 0.51 269 166 0.55 270 11 0.47 1912B 0.92 16.78 −139 53 −59 −50 0.44 190 127 0.51 268 166 0.55 272 11 0.47 1872C 2.35 1.52 −31 −55 −59 −51 0.44 31 57 0.55 220 165 0.55 260 12 0.47 1912D 3.24 0.33 −139 −55 −59 −50 0.44 30 57 0.55 220 165 0.55 261 12 0.47 1862E 2.64 0.92 −31 49 58 −52 0.45 169 127 0.72 91 91 0.53 240 17 0.47 1912F 3.66 0.16 −139 49 58 −51 0.45 169 127 0.72 91 91 0.53 242 17 0.47 1872G 3.21 0.35 −28 −52 23 38 0.42 24 53 0.74 151 94 0.75 278 89 0.45 1902H 4.05 0.08 −139 −53 23 39 0.42 23 53 0.73 151 94 0.75 279 90 0.46 1842I 3.54 0.20 −27 54 41 38 0.44 195 125 0.67 139 85 0.75 280 89 0.45 1902J 4.46 0.04 −139 54 41 39 0.44 194 125 0.67 138 86 0.75 281 89 0.46 1842K 9.22 0.00 −32 −55 14 −51 0.44 17 52 0.64 124 113 0.53 234 23 0.47 1912L 10.25 0.00 −138 −55 16 −48 0.44 16 52 0.64 127 111 0.52 237 28 0.46 1862M 9.44 0.00 −31 54 −57 55 0.44 194 127 0.49 238 159 0.78 320 83 0.50 1962N 10.23 0.00 −138 54 −57 55 0.44 195 127 0.49 237 159 0.77 321 83 0.50 1922O 10.14 0.00 −32 −54 −53 55 0.44 26 56 0.53 198 158 0.77 319 83 0.50 1952P 10.88 0.00 −138 −54 −52 54 0.44 25 56 0.53 197 157 0.77 319 83 0.50 191

4A 0.00 81.94 −31 −51 −2 −54 0.43 0 52 0.49 258 52 0.59 304 4 0.47 1914B 0.90 18.06 −137 −51 −2 −54 0.43 0 52 0.49 258 52 0.59 306 5 0.47 1864C 5.97 0.00 −32 55 3 −54 0.47 218 118 0.52 276 48 0.59 332 4 0.48 1914D 6.89 0.00 −138 56 3 −54 0.47 218 118 0.52 276 48 0.59 330 5 0.48 1864E 13.18 0.00 −33 −50 2 44 0.45 353 53 0.50 246 51 0.74 329 77 0.50 1964F 13.95 0.00 −136 −50 2 43 0.45 353 53 0.50 246 51 0.74 329 77 0.50 1924G 20.03 0.00 −32 54 6 39 0.47 226 116 0.51 267 46 0.73 332 74 0.50 1964H 20.71 0.00 −137 54 6 37 0.47 225 116 0.51 268 46 0.72 332 73 0.50 192

3A 0.00 82.34 −31 51 3 −51 0.43 179 128 0.42 94 128 0.54 252 14 0.47 1903B 0.91 17.65 −138 51 3 −50 0.43 178 128 0.42 93 128 0.54 255 15 0.47 1863C 5.77 0.00 −31 −56 −1 −51 0.47 37 62 0.49 113 129 0.54 244 17 0.47 1913D 6.68 0.00 −138 −56 −1 −50 0.47 38 62 0.49 113 128 0.54 247 17 0.47 1863E 6.01 0.00 −32 52 9 53 0.43 188 129 0.43 112 120 0.75 314 84 0.49 1953F 6.81 0.00 −137 52 9 52 0.43 187 128 0.42 113 120 0.75 313 84 0.50 1903G 9.21 0.00 −30 −55 6 42 0.46 36 61 0.52 150 108 0.74 288 86 0.45 1893H 9.98 0.00 −138 −55 6 44 0.46 36 61 0.52 148 109 0.74 290 87 0.47 185

τS: C16–C17–C20–O20;τA: C10–C1–C2–C3;τB: C5–C6–C7–C8;τC: C9–C11–C12–C13.The bold data highlight the geometrical differences with respect to the respective global minimum conformation.

1, the global minimum geometry of the tetracyclyc system(2A + 2B) accounts for about 96% of the overall population.The acetyl side chain at C17 has the two usual minimumenergy orientations with the same preference shown by com-pound1 (see2A versus2B). Though slightly bent, retropro-gesterone2 maintains a distance between the extremes ofthe molecule similar to1; in fact, the distances between thecarbonyl oxygen atoms is 11.82 and 11.67A in 1A and2A,respectively.

3.3. Conformational properties of6-dehydroprogesterone 4

The presence of the additional double bond at positionC6–C7 makes compound4 significantly more rigid than1.The inversion of A ring from the preferred 1�,2�-half-chairgeometry to the opposite half-chair is thermodynamicallymuch more disfavored; in fact,4C is less stable than4A byalmost 6 kcal/mol, though the energy barrier determined by

60 D. Colombo et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 56–62

locating the transition state of the conformational intercon-version4A → 4C is about the same (7.42 kcal/mol) as in1.B ring always assumes the 9�,10�-half-chair geometry andthe inversion of C ring becomes even more difficult than in1(4A → 4E, 13.18 kcal/mol) ensuring that the tetracyclic sys-tem is described by a single geometry without equilibrationwith other geometries. Thus, the only populated conforma-tions are4A and4B whose only difference is the acetyl sidechain at C17 which presents the two usual orientations shownin compound1 with the same preference forτS (−31◦). 4Aand4B closely resemble1A and1B in their shape and in theirdistance between the carbonyl groups.

3.4. Conformational properties of6-dehydroretroprogesterone (dydrogesterone) 3

Compound3 combines the retro-structure of2 and thedouble bond of4, the former favoring flexibility and the latterrigidity. The influence of the C6–C7 double bond largelyprevails as only two conformations of dydrogesterone3 arepopulated,3A and3B. A ring shows the same geometry as in2, i.e., the 1�,2�-half-chair, with no significant contributionto the overall population of inverted geometries at this ringbut also at the other ones. In fact, though the energy cost forthe inversion of A and C rings is lower than in4, (3A → 3C,5.77 kcal/mol and3A → 3E, 6.01 kcal/mol) it remains high.Tb aluec onlyd in atC hec nd ina

3

ts(a QCa ,a es oft ces oft signt undso on-t diedc heo rtedt hesec nt a-taHH 4a

Table 21H NMR chemical shifts (ppm) of compounds1–41H 1 2 4 3

1� 1.49 1.98 1.52 2.081� 1.80 1.52 1.78 1.482� 2.37 2.48 2.45 2.602� 2.41 2.36 2.59 2.484 5.84 5.84 5.87 5.876 – – 6.11 6.156� 2.08 2.32 – –6� 2.21 1.94 – –7 – – 5.98 6.057� 0.85 1.68 – –7� 1.60 1.39 – –8 1.31 1.60–1.62 2.02 2.289 0.75 1.45 1.06 1.6311� 1.40 1.60–1.62 1.42 1.8211� 1.25 1.60–1.62 1.20 1.6012� 1.25 1.60–1.62 1.25 1.4912� 1.94 1.76 1.95 1.8214 0.95 1.81 1.12 1.6315� 1.52 1.60–1.62 1.68 1.6815� 1.11 1.00 1.22 1.1716� 1.60 1.60–1.62 1.62 1.6116� 2.30 2.27 2.32 2.3017 2.44 2.55 2.46 2.4518 0.62 0.61 0.64 0.6319 0.98 1.22 0.97 1.1721 2.08 2.10 2.07 2.09

16�; CH319 and H2�, H6�, H14; CH321 and H12�, H12�,H17. 6-Dehydroprogesterone (4): Noe between H7 and H14,H15�, H15�; H14 and H17; CH318 and H8, H12�, H16�;CH319 and H1�, H2�, H8, H11�; CH321 and H12�, H17. 6-Dehydroretroprogesterone (3): Noe between H1� and H11�;H7 and H15�, H15�; CH318 and H8, H12�, H15�, H16�;CH319 and H1�, H2�, H12�, H14.

Table 313C NMR chemical shifts (ppm) of compounds1–413C 1 2 4 3

1 35.86 38.10 34.11 35.782 34.36 34.16 34.36 34.403 198.27 198.31 198.34 198.294 124.16 124.01 124.08 124.175 170.29 171.63 163.12 162.696 32.69 29.01 128.12 127.087 32.07 29.29 140.75 140.618 35.42 35.35 37.66 38.679 53.69 45.64 50.66 39.86

10 38.61 40.13 36.13 37.3211 21.15 22.58 20.79 20.6012 38.66 37.47 38.58 37.7313 43.85 44.22 44.64 44.2014 55.93 47.60 53.57 49.9115 24.45 24.98 23.95 25.2616 23.09 22.36 23.12 22.8617 63.43 64.45 63.19 63.221 61 92 072 6

he energy barrier for the conversion3A → 3C determinedy location of the corresponding transition state has a vomparable with the other cases (6.69 kcal/mol). Theegree of conformational freedom is the acetyl side cha17 with its two usual minimum energy orientations. Tarbonyl groups are at a distance very close to that foull the other compounds (11.74A in 3A).

.5. NMR data of compounds 1–4

Complete 1H and 13C NMR signal assignmenTables 2 and 3) of the spectra of compounds1–4 werechieved using a combination of 1D and 2D (COSY, HMnd NOESY) experiments recorded ind5-pyridine at 298 Ks this solvent gave a good spread of proton resonanc

hese steroids. Starting from the characteristic resonanhe H4, H6, H7 and H17 protons, it was possible to ashe resonances of all the other protons of the four compon the basis of their 2D spectra. The following Noe c

acts were observed in the NOESY spectra of the stuompounds which were useful for the assignment of t�r � configuration of geminal protons and also suppo

he calculated preferred conformations. In particular tontacts correspond to distances of <3A as measured ohe computed1A–4A (Fig. 1) most populated conformions of compounds1–4. Progesterone (1): Noe between H7�nd H14, H9; H14 and H17; CH318 and H8, H11�, H12�,15�, H16�; CH319 and H2�, H6�, H8, H11�; CH321 and12�, H12�, H17. Retroprogesterone (2): Noe between H1nd H6�, H17; H7� and H15�; CH318 and H12�, H15�,

8 13.39 12.72 13.23 12.09 17.08 22.63 16.17 22.10 208.28 208.24 208.14 208.1 31.32 31.31 31.33 31.3

D. Colombo et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 56–62 61

Table 4Experimental1H NMR coupling constants (Hz) of compounds1–4 in comparison with the values calculated with the electronegativity-modified Karplusrelationship

J 1 2 4 3

Calculated Experimental Calculated Experimental Calculated Experimental Calculated Experimental

1�,2� 3.9 5.2 3.6 5.2 4.6 4.9 4.2 5.61�,2� 13.1 13.6 2.8 3.1 13.3 14.3 2.3 2.01�,2� 3.3 3.2 13.5 14.7 2.3 1.7 13.3 14.71�,2� 3.4 4.9 4.1 4.9 4.2 5.2 4.6 4.94,2� – <1.0 – – – <1.0 – –4,2� – – – 1.0 – – – nd4,6� – – – 1.0 – – – nd4,6� – 2.1 – – – nd – –6,7 – – – – – 9.8 – 9.87,8 – – – – – 2.1 – 4.96,8 – – – – – 2.8 – –6�,7� 3.8 4.2 2.8 3.8 – – – –6�,7� 2.8 2.5 13.3 13.3 – – – –6�,7� 13.1 14.3 3.7 3.8 – – – –6�,7� 3.8 5.2 2.9 3.5 – – – –7�,8 12.3 11.2 1.8 2.1 – – – –7�,8 3.5 3.5 5.0 5.2 – – – –8,9 12.1 11.2 5.4 5.9 12.1 10.8 6.3 6.09,11� 3.4 4.2 1.2 <1.0 3.2 3.8 1.1 nd9,11� 12.3 11.2 6.9 6.7 12.3 12.2 7.3 nd11�,12� 3.8 nd 4.1 nd 3.9 nd 4.0 4.011�,12� 2.9 nd 2.6 nd 2.8 nd 2.7 nd11�,12� 13.2 nd 12.9 nd 13.1 nd 12.9 nd11�,12� 4.0 nd 4.6 nd 4.1 nd 4.5 nd8,14 12.1 11.2 12.0 12.6 12.0 11.6 12.1 11.014,15� 5.7 7.0 5.6 7.0 5.7 7.0 5.7 7.014,15� 11.6 12.2 11.7 11.9 11.6 12.2 11.6 11.015�,16� 11.6 9.5 11.6 nd 11.6 nd 11.6 9.015�,16� 2.2 1.8 2.2 nd 2.2 2.8 2.3 2.815�,16� 5.6 6.3 5.6 5.9 5.6 6.3 5.5 5.515�,16� 11.6 11.9 11.6 11.9 11.6 12.1 11.6 11.016�,17 8.2 9.1 8.2 9.1 8.2 9.4 8.2 9.016�,17 9.1 8.8 9.1 9.1 9.1 9.1 9.2 9.0

Even if some protons in the1H NMR spectrum resonatedas complex multiplets (7 and 5 protons at 1.55–1.70 ppmin the case of the retrosteroids2 and 3 and 3 protons at1.20–1.30 ppm in the case of4, seeTable 2) many of theseresulted well resolved signals the coupling of which could bemeasured. The obtained values are reported inTable 4in com-parison with the calculated constants of compounds1–4. Foreach populated conformer, the1H vicinal coupling constantswere calculated with the electronegativity-modified Karplusrelationship[17] and were weighted averaged on the basisof the population percentages. The close agreement of theexperimental with the calculated values supports the confor-mational preferences of the four steroids determined throughthe calculations.

4. Discussion

The modeling study here reported, supported by the NMRexperiments, allows to define the exact role of the C9 and C10configurational inversion as well as that of the C6–C7 unsat-

uration. The C9 and C10 configurational inversion modifiesthe properties of the steroidal skeleton introducing some dif-ferences in the preferred geometry, in particular, of A andB rings. For example, A ring prefers the 1�,2�-half-chair inthe normal steroids and the opposite 1�,2�-half-chair geome-try in the retrosteroids (Table 1). It has been observed[18,19]that A ring inversion induced by a double bond at the C9–C10position gives structures which often exhibit a very high affin-ity for the progesterone receptor. This inversion occurs also inthe retrosteroids2 and3 and they both have a progestationalactivity higher than the corresponding normal steroids1 and4. Though2 and 3 have a bent shape at the B/C junction,A ring inversion restores a high degree of planarity so thatthe overall shape of the retro and the normal steroids is veryclose. This is evidenced by the distances between the carbonylgroups, about the same in all the cases, and by the superim-position of1A with 2A and3A with 4A (Fig. 2) obtainedthrough the best rms fit of the atoms at the two extremes ofthe molecules.

The C6–C7 double bond introduces rigidity in both thenormal and the retro skeletons so that the two 6-dehydro

62 D. Colombo et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 56–62

Fig. 2. Superimposition of the preferred conformations of compounds1 vs.2 and3 vs.4.

steroids3 and4 are completely described by only one geome-try of the tetracyclic system the only degree of conformationalfreedom of the molecules being rotation around the C17–C20bond. On the contrary, in steroids1 and2, besides the largelypreferred global minimum conformation, are accessible otherconformations deriving from the inversion of one or morehexacyclic rings from the chair (B, C rings) or half-chair (Aring) to the twisted-boat or to the opposite half-chair geom-etry, respectively.

In conclusion, the most active compound, dydrogesterone3, takes advantage both from its retro-structure, which forcesA ring towards the 1�,2�-half-chair geometry, and from thepresence of the C6–C7 double bond, which constricts themolecule into a rigid conformation probably suitable for theinteraction with the progesterone receptor. The higher rigidityof 3 also positively influences its selectivity while proges-terone1, less selective, exists in different conformations andmay adapt more easily to the requisites of different receptors.

Acknowledgements

This work was financially supported by Universita diMilano (FIRST funds) and Universita di Pavia (FAR funds).We warmly thank professor Fiamma Ronchetti for very help-ful discussions.

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