Patrizia
Ferraboschi
a,
Laura
Legnani
ab,
Giuseppe
Celasco
c,
Luigi
Moro
c,
Laura
Ragonesi
c and
Diego
Colombo
*a
aDipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università di Milano, Via Saldini 50, 20133 Milano, Italy. E-mail: diego.colombo@unimi.it; Fax: +39-0250316036; Tel: +39-0250316039
bDipartimento di Chimica – Università degli Studi di Pavia, Via Taramelli 12, 27100 Pavia, Italy
cCosmo Research & Development S.p.A., Via C. Colombo 1, 20020 Lainate, MI, Italy
First published on 4th April 2014
Cortexolone-17α-propionate is a topical antiandrogen under investigation for the treatment of androgen-related skin disorders. A full conformational characterization was realized, in comparison with other steroidal androgens and antiandrogens, by means of theoretical calculations at the B3LYP/6-31G(d) level supported by high-field NMR analyses. All of the studied molecules showed a good overlay; nevertheless, the different functional groups present in the skeleton of the molecules drive the individual biological profile.
The absence of systemic antiandrogenic effects could be explained considering that the propionate, after percutaneous application, is quickly hydrolyzed by the skin and plasma esterases into the inactive parent cortexolone (3) getting through the 21-propionate 5.
The topical activity of compound 4 is higher than that of finasteride 6 and about equivalent to that of cyproterone acetate 7.
Taking into account the topical activity of propionate 4 coupled with the lack of systemic activity, we planned to compare this 17α-ester with the well-known antiandrogen cyproterone acetate 7 and with the natural androgens, testosterone 1 and dihydrotestosterone 2, from a conformational point of view.
In fact, the conformation of a biologically active compound plays a central role when it interacts with the target, for example, a receptor or an enzyme; among the possible conformations only one could be able to stimulate the biological response as we also reported in a previous work.11 The choice of the compounds to be compared with propionate 4 was driven by its antiandrogenic activity. In fact testosterone (1) and dihydrotestosterone (2) are antagonized both by compounds 4 and 7 with the same action mechanism that does not implicate the interference with the 5α-reductase. The conformational characterization was realized by means of theoretical calculations, validated by complete assignment of the 1H and 13C NMR signals, as in the case of our previous studies of steroidal compounds.11
Topical treatment (acetone 0.05 mL) | Daily doseb | Flank organ inhibitionc (%) |
---|---|---|
a The local antiandrogenic activity of 4 and other tested compounds 3, 6, and 7 was expressed as the percentage inhibition of the flank organ enlargement induced by the topical application of testosterone propionate (TP) alone. b μg per animal, antiandrogen + androgen. c *P < 0.05, **P < 0.01. | ||
Cortexolone (3) + TP | 400 + 4 | 0 |
Finasteride (6) + TP | 400 + 4 | 71* |
Cyproterone acetate (7) + TP | 400 + 4 | 93** |
Cortexolone-17α-propionate (4) + TP | 100 + 4 | 40* |
Cortexolone-17α-propionate (4) + TP | 200 + 4 | 78* |
Cortexolone-17α-propionate (4) + TP | 400 + 4 | 84** |
As concerns the mechanism of action, compound 4, compared to finasteride (6) the well-known inhibitor of 5α-reductase, did not inhibit the conversion of testosterone (1) to DHT (2) in reconstructed human epidermis (Cosmo R & D personal communication), thus resulting in the absence of inhibitory activity on the 5α-reductase, as shown by the studied [14C]-testosterone metabolism after 24 h transepidermal diffusion (Fig. 2).
![]() | ||
Fig. 2 [14C]-testosterone (T, 1) metabolism and DHT (2) production in presence of cortexolone-17α-propionate (4) or finasteride (6). |
Additional experiments (Cosmo R & D personal communication) showed that in the binding affinity test to the androgen-receptor of human prostate cancer cells, compound 4 inhibited the specific binding of [3H] methyltrienolone (R1881) to the androgen receptor with a Ki value of 4.0 × 10−8, and an IC50 value of 5.0 × 10−8 M. As a consequence, compound 4 should be considered as an antiandrogen acting at the androgen-receptor level.
Incubation time | Cortexolone-17α-propionate (4) (%) | Cortexolone (3) (%) |
---|---|---|
0 | 100 | 0 |
5 min | 95–90 | 5–10 |
15 min | 95–90 | 5–10 |
30 min | 90–80 | 10–20 |
1 h | 80–60 | 20–40 |
2 h | 50 | 50 |
4 h | 40–20 | 60–80 |
8 h | 10–0 | 90–100 |
An analogous metabolic profile of 4 was observed with incubation in human plasma (Table 3).
Incubation time | Cortexolone-17α-propionate (4) (%) | Cortexolone-21-propionate (5) (%) | Cortexolone (3) (%) |
---|---|---|---|
0 | 99.6 | 0.5 | 0.0 |
30 min | 93.2 | 6.5 | 0.4 |
1 h | 85.2 | 13.2 | 1.7 |
2 h | 67.2 | 25.3 | 7.5 |
4 h | 30.3 | 33.8 | 35.9 |
6 h | 11.3 | 23.8 | 64.9 |
In rat skin homogenate the metabolic transformation of 4 to cortexolone (3) reached a peak (40–44.7%) within 8–16 h, and remained stable during the remnant incubation period of up to 24 h (Table 4).
Incubation time | Cortexolone-17α-propionate (4) (%) | Cortexolone-21-propionate (5) (%) | Cortexolone (3) (%) |
---|---|---|---|
0 | 99 | 0.5 | 0 |
5 min | 99 | 0.5 | 0 |
15 min | 99 | 0.5 | 0 |
30 min | 98.5 | 0.5 | 0.5 |
1 h | 89.5 | 5 | 5 |
2 h | 69.5 | 15 | 15 |
4 h | 59.5 | 10 | 30 |
8 h | 49.5 | 10 | 40 |
16 h | 44.75 | 10 | 44.75 |
24 h | 44.75 | 10 | 44.75 |
Compound | Flank organ inhibitiona (%) |
---|---|
a Ability of the tested steroids (400 μg) to inhibit the enlargement of the hamster's organ flank produced by the administration of 4 μg of testosterone propionate. | |
Cortexolone-21-propionate (5) | 29 |
Cortexolone-17α,21-dipropionate (8) | 57 |
9,11-Dehydrocortexolone-17α-butyrate (9)12 | 85 |
Differently from the 17α-monoesters of cortexolone, 9,11-dehydrocortexolone-17α-butyrate (9) was found to show systemic activity in the rat after subcutaneous injection.12
Compound 9 was also discovered to be a potent inhibitor of gonadotropin hypersecretion, thus mimicking the activity profile of cyproterone acetate (7), which blocks the androgen-receptor interaction and simultaneously reduces serum testosterone through its antigonadotropic action.13,14 The presence of a double bond at position 9,11 of the cortexolone, modifying the spatial conformation of the steroids rings, could be responsible for the systemic and increased topical activity of 9.12
E rel (kcal mol−1) | % | τ A (°) | τ B (°) | τ C (°) | τ 1 (°) |
τ
1′![]() |
τ 2 (°) | τ 3 (°) | Ring puckering coordinates | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
A ring | B ring | C ring | D ring | |||||||||||||||||
Q | ϕ 2 | θ | Q | ϕ 2 | θ | Q | ϕ 2 | θ | q 2 | ϕ 2 | ||||||||||
a τ A: C10–C1–C2–C3. b τ B: C5–C6–C7–C8. c τ C: C9–C11–C12–C13. d τ 1: C16–C17–O–H for 1 and 2, C16–C17–C20–C21 for 3, 4, 7–9. e τ 1′: C17–C20–C21–O. f τ 2: C16–C17–O–H for 3, C16–C17–O–C17′ for 4, 7–9. g τ 3: C20–C21–O–C22. | ||||||||||||||||||||
1A | 0.00 | 59.8 | −54 | 54 | −54 | 172 | 0.44 | 16 | 54 | 0.54 | 167 | 7 | 0.54 | 272 | 6 | 0.46 | 187 | |||
1Ab | 0.27 | 37.7 | −54 | 54 | −54 | 64 | 0.44 | 16 | 54 | 0.54 | 167 | 7 | 0.57 | 271 | 6 | 0.46 | 187 | |||
1B | 1.88 | 2.5 | 55 | 55 | −54 | 172 | 0.44 | 203 | 125 | 0.58 | 347 | 5 | 0.58 | 294 | 5 | 0.46 | 187 | |||
1C | 6.05 | 0.0 | −48 | −51 | −54 | 172 | 0.45 | 347 | 54 | 0.72 | 261 | 84 | 0.59 | 335 | 5 | 0.47 | 188 | |||
1D | 11.13 | 0.0 | −54 | 53 | 45 | 174 | 0.45 | 12 | 55 | 0.56 | 12 | 55 | 0.73 | 324 | 79 | 0.49 | 193 | |||
2A | 0.00 | 62.8 | −51 | 54 | −54 | 173 | — | — | — | 0.54 | 285 | 10 | 0.57 | 320 | 4 | 0.57 | 275 | 5 | 0.47 | 188 |
2Ab | 0.31 | 36.9 | −51 | 54 | −54 | 64 | — | — | — | 0.54 | 285 | 10 | 0.58 | 319 | 4 | 0.57 | 275 | 5 | 0.46 | 187 |
2B | 3.15 | 0.3 | 28 | 55 | −54 | 173 | — | — | — | 0.77 | 265 | 85 | 0.56 | 210 | 4 | 0.57 | 276 | 5 | 0.46 | 188 |
2C | 11.83 | 0.0 | −45 | −42 | −54 | 173 | — | — | — | 0.56 | 270 | 19 | 0.71 | 274 | 77 | 0.60 | 344 | 5 | 0.47 | 188 |
2D | 11.50 | 0.0 | −49 | 55 | 45 | 174 | — | — | — | 0.54 | 278 | 12 | 0.59 | 216 | 5 | 0.73 | 215 | 78 | 0.50 | 193 |
3A | 0.63 | 24.8 | −54 | 54 | −54 | 146 | 175 | −45 | — | 0.44 | 17 | 54 | 0.54 | 165 | 6 | 0.57 | 270 | 4 | 0.48 | 187 |
3Ab | 0.00 | 71.7 | −54 | 54 | −54 | −24 | −169 | −159 | — | 0.44 | 17 | 54 | 0.54 | 169 | 6 | 0.57 | 271 | 5 | 0.48 | 188 |
3B | 2.58 | 0.9 | 55 | 55 | −55 | 147 | 175 | −44 | — | 0.44 | 203 | 125 | 0.58 | 350 | 5 | 0.58 | 302 | 3 | 0.48 | 188 |
3Bb | 1.96 | 2.6 | 55 | 55 | −54 | −24 | −169 | −158 | — | 0.44 | 203 | 125 | 0.58 | 349 | 5 | 0.58 | 299 | 4 | 0.48 | 188 |
3Cb | 6.14 | 0.0 | −48 | −51 | −54 | −24 | −169 | −158 | — | 0.45 | 347 | 54 | 0.72 | 261 | 84 | 0.59 | 339 | 5 | 0.48 | 189 |
3Db | 10.55 | 0.0 | −54 | 53 | 45 | −25 | −169 | −156 | — | 0.44 | 11 | 54 | 0.56 | 182 | 13 | 0.74 | 324 | 78 | 0.51 | 193 |
4A | 0.00 | 66.0 | −54 | 54 | −55 | 156 | 163 | −65 | — | 0.44 | 17 | 54 | 0.54 | 163 | 6 | 0.57 | 271 | 3 | 0.47 | 190 |
4Ab | 0.46 | 30.3 | −54 | 54 | −54 | −13 | −172 | −75 | — | 0.44 | 17 | 54 | 0.54 | 166 | 7 | 0.57 | 271 | 5 | 0.46 | 188 |
4B | 1.93 | 2.6 | 55 | 54 | −55 | 156 | 163 | −65 | — | 0.44 | 202 | 125 | 0.58 | 345 | 5 | 0.58 | 298 | 3 | 0.47 | 190 |
4Bb | 2.44 | 1.1 | 55 | 55 | −54 | −13 | −172 | −75 | — | 0.44 | 203 | 125 | 0.58 | 352 | 5 | 0.58 | 301 | 4 | 0.46 | 188 |
4C | 6.20 | 0.0 | −49 | −51 | −55 | 156 | 164 | −65 | — | 0.45 | 348 | 53 | 0.72 | 261 | 84 | 0.59 | 353 | 4 | 0.48 | 192 |
4D | 10.59 | 0.0 | −54 | 53 | 45 | 157 | 168 | −64 | — | 0.44 | 13 | 55 | 0.56 | 183 | 12 | 0.73 | 325 | 78 | 0.49 | 199 |
8A | 1.29 | 9.8 | −54 | 54 | −54 | 155 | 150 | −65 | 95 | 0.44 | 17 | 54 | 0.54 | 166 | 6 | 0.57 | 268 | 4 | 0.46 | 190 |
8Ab | 0.00 | 85.8 | −54 | 54 | −53 | −11 | −179 | −75 | 77 | 0.44 | 16 | 54 | 0.54 | 165 | 6 | 0.57 | 278 | 5 | 0.46 | 185 |
8B | 3.17 | 0.4 | 55 | 55 | −55 | 156 | 149 | −66 | 96 | 0.44 | 203 | 125 | 0.58 | 353 | 5 | 0.58 | 312 | 3 | 0.47 | 191 |
8Bb | 1.81 | 4.0 | 55 | 54 | −53 | −11 | −179 | −75 | 77 | 0.44 | 202 | 125 | 0.58 | 346 | 5 | 0.58 | 301 | 4 | 0.47 | 185 |
8Cb | 6.10 | 0.00 | −48 | −51 | −54 | −11 | −179 | −75 | 77 | 0.45 | 347 | 54 | 0.72 | 261 | 84 | 0.59 | 344 | 5 | 0.47 | 187 |
8Db | 10.25 | 0.00 | −54 | 53 | 43 | −12 | −179 | −74 | 77 | 0.46 | 22 | 49 | 0.56 | 183 | 13 | 0.73 | 323 | 78 | 0.49 | 192 |
9A | 0.00 | 55.0 | −55 | 56 | −16 | 154 | 162 | −66 | — | 0.44 | 15 | 54 | 0.50 | 117 | 12 | 0.51 | 266 | 52 | 0.45 | 187 |
9Ab | 0.25 | 36.0 | −55 | 56 | −15 | −13 | −172 | −76 | — | 0.44 | 14 | 54 | 0.50 | 116 | 12 | 0.51 | 267 | 52 | 0.45 | 183 |
9B | 1.34 | 5.7 | 56 | 54 | −16 | 156 | 163 | −65 | — | 0.45 | 203 | 125 | 0.55 | 17 | 10 | 0.51 | 267 | 51 | 0.45 | 184 |
9Bb | 1.68 | 3.2 | 56 | 54 | −16 | −13 | −172 | −76 | — | 0.45 | 203 | 125 | 0.55 | 20 | 10 | 0.51 | 267 | 51 | 0.44 | 183 |
9C | 3.77 | 0.1 | −50 | −57 | −14 | 157 | 162 | −76 | — | 0.46 | 348 | 54 | 0.70 | 264 | 89 | 0.50 | 272 | 49 | 0.45 | 186 |
7A | 0.00 | 82.0 | −6 | −1 | −54 | 156 | — | −67 | — | 0.30 | 301 | 81 | 0.49 | 259 | 51 | 0.58 | 316 | 3 | 0.47 | 191 |
7Ab | 0.90 | 18.0 | −6 | −1 | −54 | −10 | — | −76 | — | 0.30 | 301 | 81 | 0.49 | 259 | 51 | 0.59 | 314 | 4 | 0.46 | 188 |
Some short contacts characterize the conformational preferences of the rings of 2: H-2ax/CH3-10 (2.81 Å), and H-4ax/CH3-10 (2.76 Å) for the A ring; H-6ax/CH3-10 (2.72 Å), H-8ax/CH3-10 (2.86 Å) for the B-ring conformation; H-11ax/CH3-10 (2.73 Å), H-11ax/CH3-13 (2.76 Å), and H-8ax/CH3-13 (2.78 Å) for the C ring; H-15ax/CH3-13 (2.90 Å) for ring D.
The energy profiles for rotation around the C17–C20, and C17–O single bonds, defined by τ1 and τ2, were obtained and the preferred orientations determined. The C17–C20 bond showed a quite balanced distribution of its possible orientations, with the presence, for all compounds, of two significantly populated geometries that present τ1 ≈ 160, and τ1 ≈ −10, respectively. For 3 and 8 conformation Ab, with τ1 ≈ −10, is favoured by 0.63 and 1.29 kcal mol−1, respectively, whereas 4 and 9 prefer the other orientation by 0.46 and 0.25 kcal mol−1, respectively. Concerning the C17–O bond, in the case of 4, 8, and 9, a significant preference was observed for the orientation characterized by τ2 ≈ −60°, with the other higher in energy by 4–6 kcal mol−1, whereas in 3 the hydroxyl group bonded at C17 shows two orientations related to the value of τ1, being preferred τ2 = −45 for τ1 = 146, and τ2 = −159 for τ1 = −24. In compound 8, the hydroxyl at C21, present in 3, 4, and 9, is esterified, with the generation of a second propionate group. Considering the oxoethyl propionate bonded at C17 the torsional angle τ3 describes the relative orientation of the two carbonyl groups of the chain. The second one is perpendicular to the first (τ3 ≈ 90°), whatever the orientation of this latter (see τ1).
A careful analysis of the conformational freedom of the tetracyclic skeleton allowed us to determine the facility of inversion of the three hexacyclic rings. The A ring inversion from the 1α,2β-half-chair to the 1β,2α-half-chair conformation (3, 4, 8, 9A → 3, 4, 8, 9B) is the easiest among all the possible ring inversions. In the case of compounds 3, 4, and 8, the A ring inversion brings about a conformation less stable by about 2 kcal mol−1. For compound 9 the same inversion is easier, giving the conformation 9B with an energy value of 1.34 kcal mol−1. So the percentage contribution of the 1β,2α-half-chair conformation to the overall population is double in the case of 9 with respect to the others, although the 1α,2β-half-chair conformation remains widely preferred.
As regards B ring inversion, the obtained conformations C, presenting a relative energy of about 4–6 kcal mol−1, respectively, do not give any contribution to the overall population. The ring C inversion of 9 is not possible because of the presence of the double bond, whereas for 3, 4 and 8 a conformation (D) is obtained that is higher in energy than the global minimum by more than 10 kcal mol−1.
In 3, 4, 8 and 9 the preference of ring A for the 1α,2β-half-chair conformation is characterized by the short contact H-2ax/CH3-19 (2.90 Å). The contacts H-6ax/CH3-19 (2.91 Å), and H-8ax/CH3-19 (2.87 Å) confirm the B ring conformation; contacts H-8ax/CH3-18 (2.74 Å), H-11ax/CH3-19 (2.70 Å), and H-11ax/CH3-18 (2.73 Å) assure the C ring geometry, whereas contact H-15ax/CH3-18 (2.90 Å) gives the D ring conformation.
Finally, the different orientations of τ1 could be verified through contacts H-21b/H-12eq (2.48, 2.29, 2.36 Å, respectively, for 3, 4, and 8) for conformation A; H-21a,b/CH3-18 (2.63, 2.82, 2.90 Å), H-21a/H-16 (2.42, 2.25, 2.27 Å), and H-21b/H-16 (2.37, 2.43, 2.46 Å) for conformation Ab.
Analogously, conformation A of compound 9 presents the contacts: H-2ax/CH3-19 (2.85 Å) for ring A; H-6ax/CH3-19 (2.94 Å) for ring B; H-21a/H-12eq (2.39 Å) for τ1 = 156. The second orientation of τ1 could be verified through contacts: H-21b/CH3-18 (2.88 Å), H-21b/H-16 (2.24 Å), and H-21a/H-16 (2.48 Å).
Compound 7 shows a rigid structure and the only degree of conformational freedom is the inversion of ring C. Rings A and B could not be inverted because of the presence of the cyclopropane ring and the double bond, respectively. Nevertheless, the C ring inversion gave conformations with a relative energy of about 13 kcal mol−1, giving no contribution to the overall population. So, only two geometries, 7A and 7Ab, are populated (Fig. 5). Conformation A of compound 7 presents the following contacts: H-11β/CH3-18 (2.24 Å), H-11β/CH3-19 (2.29 Å), H-8/CH3-19 (2.76 Å), and H-9/CHa-cPr (2.66 Å).
On the basis of crystallographic studies15 performed on dihydrotestosterone (2) complexed with the ligand-binding domain of the wild-type androgen receptor, both the carbonyl oxygen atom bonded at C3 and the hydroxyl group at C17 of this molecule actively contribute to the stabilization of the obtained complex. The A ring conformation influences the orientation of the carbonyl group that deeply affects the binding of the entire molecule.
A docking study performed on 7,16 into the homology model for the glucocorticoid receptor ligand binding domain, revealed that in the active site it assumes conformation A.
The superimposition of the heavy atoms of the tetracyclic system of the preferred conformations of compounds 3, 4 and 7–9 (Fig. 6) gave evidence that the presence of the hydroxyl group or the ester chain, bonded to C21 in compounds 3, 4, 8 and 9, does not affect the orientation of the substituent at C17.
Compounds 3, 4, 8 and 9 show a very good overlay, also concerning the O3 atoms that are perfectly coincident, in spite of the presence in 9 of a double bond on ring C. Conversely, the overlay shows that in 7 the O3 atom is differently oriented and diverges with the distance d(O–O) ≈ 1.0 Å.
1H | 3 | 4 | 7 | 8 | 9 |
---|---|---|---|---|---|
1 | 1.70 | ||||
1α | 1.66 | 1.71 | — | 1.70 | 2.06–2.17 |
1β | 2.00 | 2.02 | — | 2.02 | 2.06–2.17 |
2 | 2.00 | ||||
2α | 2.32 | 2.32 | — | 2.34 | 2.43–2.50 |
2β | 2.39 | 2.41 | — | 2.40 | 2.43–2.50 |
CHa (cPr) | — | — | 0.86 | — | — |
CHb (cPr) | — | — | 1.26 | — | — |
4 | 5.70 | 5.72 | 6.16 | 5.72 | 5.74 |
6α | 2.26 | 2.27 | — | 2.27 | 2.35 |
6β | 2.38 | 2.38 | — | 2.38 | 2.56 |
7 | — | — | 6.20 | — | — |
7α | 1.08 | 1.10 | — | 1.08 | 1.16 |
7β | 1.85 | 1.85 | — | 1.84 | 2.01 |
8 | 1.59 | 1.60 | 2.31 | 1.62 | 2.22 |
9 | 0.95 | 1.00 | 1.45 | 0.99 | — |
11 | — | — | — | — | 5.52 |
11α | 1.62 | 1.65 | 1.94 | 1.65 | — |
11β | 1.38 | 1.40 | 1.55 | 1.45 | — |
12α | 1.72 | 1.89 | 2.03 | 1.88 | 2.75 |
12β | 1.40 | 1.54 | 1.61 | 1.74 | 1.76 |
14 | 1.70 | 1.67 | 1.96 | 1.67 | 1.85 |
15α | 1.80 | 1.76 | 1.89 | 1.72 | 1.94 |
15β | 1.37 | 1.35 | 1.44 | 1.34 | 1.45 |
16α | 1.57 | 1.85 | 1.82 | 1.84 | 1.97 |
16β | 2.66 | 2.81 | 2.98 | 2.82 | 2.80 |
17 (OCOCH2) | — | 2.34 | — | 2.35 | 2.28 |
17 (OCOCH2CH3) | — | 1.12 | — | 1.13 | — |
17 (OCOCH2CH2) | — | — | — | — | 1.62 |
17 (OCOCH2CH2CH3) | — | — | — | — | 0.93 |
17 (OCOCH3) | — | — | 2.09 | — | — |
17 OH | 2.45 | — | — | — | — |
18 (CH3) | 0.68 | 0.66 | 0.71 | 0.74 | 0.61 |
19 (CH3) | 1.16 | 1.17 | 1.21 | 1.16 | 1.32 |
21 (CH3) | — | — | 2.04 | — | — |
21a | 4.28 | 4.21 | — | 4.59 | 4.24 |
21b | 4.64 | 4.26 | — | 4.87 | 4.28 |
21 (OCOCH2) | — | — | — | 2.45 | — |
21 (OCOCH2CH3) | — | — | — | 1.15 | — |
21 OH | 3.09 | 3.03 | — | — | 3.04 |
13C | 3 | 4 | 7 | 8 | 9 |
---|---|---|---|---|---|
1 | 35.68 | 35.70 | 26.07 | 35.67 | 33.83 |
2 | 33.87 | 33.91 | 25.22 | 33.92 | 34.22 |
CH2 (cPr) | — | — | 12.29 | — | — |
3 | 199.58 | 199.26 | 197.94 | 199.40 | 199.05 |
(199.04) | |||||
4 | 123.94 | 124.07 | 120.51 | 123.98 | 124.17 |
5 | 170.93 | 170.33 | 155.22 | 170.61 | 169.01 |
6 | 32.73 | 32.65 | 130.23 | 32.69 | 32.70 |
7 | 31.98 | 31.91 | 136.51 | 31.91 | 32.13 |
8 | 35.59 | 35.57 | 38.33 | 35.57 | 37.48 |
9 | 53.28 | 53.16 | 47.70 | 53.14 | 144.20 |
10 | 38.54 | 38.53 | 38.70 | 38.54 | 40.98 |
11 | 20.52 | 20.50 | 20.78 | 20.57 | 118.25 |
12 | 30.08 | 30.46 | 31.02 | 30.24 | 32.35 |
13 | 48.57 | 47.72 | 47.24 | 47.79 | 46.28 |
14 | 50.28 | 50.93 | 48.77 | 50.94 | 48.16 |
15 | 23.70 | 23.89 | 23.23 | 23.77 | 24.65 |
16 | 34.52 | 30.77 | 30.30 | 30.78 | 30.52 |
17 | 89.00 | 93.77 | 96.18 | 94.86 | 93.33 |
17 (OCO) | — | 174.05 | 170.55 | 174.20 | 173.24 |
(174.02) | |||||
17 (OCOCH3) | — | — | 21.17 | — | — |
17 (OCOCH2) | — | 27.77 | — | 27.10 | 36.24 |
(27.90) | |||||
17 (OCOCH2CH3) | — | 8.85 | — | 8.97 | — |
(8.90) | |||||
17 (OCOCH2CH2) | — | — | — | — | 18.31 |
17 (OCOCH2 CH2CH3) | — | — | — | — | 13.57 |
18 | 14.99 | 14.29 | 14.19 | 13.75 | 14.31 |
19 | 17.36 | 17.39 | 22.83 | 17.35 | 26.20 |
20 | 212.33 | 206.21 | 203.61 | 199.40 | 206.22 |
(199.04) | |||||
21 | 67.42 | 66.93 | 26.43 | 66.88 | 66.91 |
21 (OCO) | — | — | — | 174.20 | — |
(174.02) | |||||
21 (OCOCH2) | — | — | — | 27.10 | — |
(27.90) | |||||
21 (OCOCH2CH3) | — | — | — | 8.97 | — |
(8.90) |
J | 3 | 3 | 4 | 4 | 7 | 7 | 8 | 8 | 9 | 9 |
---|---|---|---|---|---|---|---|---|---|---|
Exp | Calc | Exp | Calc | Exp | Calc | Exp | Calc | Exp | Calc | |
1α,1β | 13.4 | 13.4 | — | 13.4 | n.d. | |||||
1α,2α | 4.5 | 4.0 | 5.0 | 3.9 | — | n.d. | 3.9 | n.d. | 3.9 | |
1α,2β | n.d | 13.5 | n.d | 13.5 | — | n.d. | 13.6 | n.d. | 13.6 | |
1β,2α | 3.2 | 2.9 | 3.3 | 2.9 | — | 2.7 | 2.9 | n.d. | 2.9 | |
1β,2β | 5.0 | 3.4 | 5.0 | 3.4 | — | 4.8 | 3.4 | n.d. | 3.5 | |
2β,2α | 16.7 | 16.7 | — | n.d. | n.d. | |||||
1,2 | — | — | 7.9 | 10.5 | — | — | ||||
1,CHa (cPr) | — | — | 6.4 | 9.5 | — | — | ||||
1,CHb (cPr) | — | — | 7.9 | 10.3 | — | — | ||||
2,CHa (cPr) | — | — | 4.5 | 8.2 | — | — | ||||
2,CHb (cPr) | — | — | 8.9 | 10.4 | — | — | ||||
CHa,CHb(cPr) | — | — | 4.8 | — | — | |||||
6α,6β | 14.6 | 14.6 | — | 14.5 | 14.4 | |||||
6α,7α | 4.3 | 3.7 | 4.2 | 3.7 | — | 4.0 | 3.7 | 3.9 | 3.3 | |
6α,7β | 2.4 | 2.9 | 2.4 | 2.9 | — | 2.4 | 2.9 | 2.6 | 3.1 | |
6β,4 | n.d. | n.d | — | n.d. | 1.9 | |||||
6β,7α | 12.0 | 13.2 | 11.5 | 13.3 | — | 12.0 | 13.3 | 14.4 | 13.4 | |
6β,7β | n.d. | 3.8 | 5.0 | 3.8 | — | n.d. | 3.8 | 4.8 | 3.5 | |
7α,7β | 13.0 | 13.7 | — | 14.0 | 12.4 | |||||
7α,8 | 12.0 | 12.3 | 12.5 | 12.3 | — | 12.0 | 12.4 | 12.5 | 12.4 | |
7β,8 | n.d. | 3.4 | 3.3 | 3.2 | — | 3.3 | 3.2 | 4.7 | 3.2 | |
7,8 | — | — | 2.0 | 1.0 | — | — | ||||
8,9 | 10.8 | 12.1 | 11.0 | 12.3 | 9.8 | 12.9 | 10.8 | 12.9 | — | |
8,14 | n.d. | 12.1 | 11.0 | 12.3 | 10.0 | 10.8 | 10.8 | |||
8,12α | — | — | — | — | 2.9 | |||||
8,12β | — | — | — | — | 2.0 | |||||
11,8 | — | — | — | — | 2.0 | |||||
11,12α | — | — | — | — | 2.9 | 2.0 | ||||
11,12β | — | — | — | — | 5.9 | 6.4 | ||||
9,11α | 4.2 | 3.4 | 4.0 | 3.0 | 4.1 | — | ||||
9,11β | 11.8 | 12.3 | 12.5 | 12.8 | 13.0 | — | ||||
11α,12α | n.d. | 3.8 | 4.3 | 3.8 | n.d. | 4.3 | 4.0 | — | ||
11α,12β | n.d. | 2.8 | 2.9 | 2.9 | 2.7 | n.d. | 2.6 | — | ||
11β,12α | n.d. | 13.2 | 13.0 | 13.2 | 12.8 | 13.2 | 13.1 | — | ||
11β,12β | n.d. | 4.1 | 4.2 | 4.0 | 4.0 | 4.2 | 4.4 | — | ||
11α,11β | n.d. | 13.4 | 12.8 | 13.2 | — | |||||
12α,12β | n.d. | 13.0 | 12.6 | 13.0 | 17.0 | |||||
14,15α | n.d. | 5.8 | 7.0 | 5.4 | n.d. | n.d. | 7.7 | 6.1 | ||
14,15β | n.d. | 11.6 | 11.8 | 11.8 | 11.0 | 11.3 | 11.0 | 11.3 | ||
15α,16α | 9.4 | 11.0 | 9.4 | 11.8 | 9.2 | n.d. | 12.0 | 9.4 | 11.0 | |
15α,16β | 3.0 | 2.3 | 2.6 | 2.1 | 2.4 | 2.5 | 2.6 | 3.0 | 2.7 | |
15α,15β | n.d. | 11.8 | 11.6 | 11.3 | 17.0 | |||||
15β,16α | 6.2 | 5.4 | 6.5 | 5.7 | 6.0 | 6.5 | 4.7 | 5.0 | 4.9 | |
15β,16β | 11.5 | 12.0 | 11.8 | 11.8 | 10.2 | 11.3 | 12.0 | 12.5 | 12.0 | |
16α,16β | 14.8 | 16.0 | 15.7 | 15.7 | 15.2 | |||||
21a,21b | 19.8 | 18.2 | — | 16.5 | 18.3 | |||||
21a,OH | 4.5 | 4.9 | — | — | 4.9 | |||||
21b,OH | 4.5 | 4.9 | — | — | 4.8 |
These nOe data, such as the experimental values of the 1H vicinal coupling constants, supported the calculated preferred conformations. In particular, almost all these contacts correspond to distances of <3 Å as measured on the computed (Fig. 4 and 5) most populated conformations of compounds 3, 4 and 7–9.
In the present work a conformational comparison of 4 with testosterone (1), active testosterone metabolite DHT (2) and antiandrogen cyproterone acetate (7) (similar to 4 for the action mechanism direct on the androgen receptor) was carried out by means of theoretical calculations, supported by their complete high-field NMR characterization. In addition, the comparison was extended to the related compounds cortexolone (3), devoid of activity, cortexolone-17α,21-dipropionate (8), less active, and Δ9-butyrate 9, active both topically and systemically.
The conformational characterization showed that all compounds are similar (see Fig. 6), minor differences being observed: cyproterone acetate (7) has the 3-carbonyl group differently oriented and the presence of the 9,11-double bond hampers the C ring inversion in compound 9. However the 3-carbonyl group orientation (7vs.4, Table 1) and the conformation of the C ring (9vs.4, Tables 1 and 5) do not influence the extent of the local antiandrogenic activity of the tested compounds. By contrast, the presence of the 17α-ester group, which was always oriented in the same way from the conformational study, seems to be mandatory for a good inhibitory activity (3vs.4, Table 1). In conclusion, the cortexolone series compounds 3–5 and 8, cyproterone acetate (7), and Δ9-17α-butyrate 9 share the same skeleton conformation but show different antiandrogenic activities due to the presence of an acyl chain linked to the 17α-hydroxyl functionality. The same skeleton conformation of the examined compounds is consistent with the capability of each of them to interact, even with different outcomes, with the androgen receptors. Furthermore, the absence of systemic activity of 4 could be explained by its metabolic fate, i.e. the esterases-catalyzed hydrolysis of the acyl chain, after the rapid migration from the 17- to the 21-position.
Std | M | RT (min) | m/z |
---|---|---|---|
3 | 346.21 | 3.48–3.89 | 345.0 [M − 1], 691.3 [2M − 1] |
4 | 402.24 | 5.38–5.67 | 803.1 [2M − 1] |
5 | 402.24 | 7.05–7.59 | 803.2 [2M − 1] |
This journal is © The Royal Society of Chemistry 2014 |