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Synthesis and GABAA receptor activity of A-homo analogues of neuroactive steroids
European Journal of Medicinal Chemistry 45 (2010) 3063e3069
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Original article
Synthesis and GABAA receptor activity of A-homo analogues of neuroactive steroids María V. Dansey a, Pablo H. Di Chenna a, Adriana S. Veleiro a, Zdena Kristofíková b, Hana Chodounska c, Alexander Kasal c, Gerardo Burton a, * a
Departamento de Química Orgánica and UMYMFOR (CONICET-FCEN), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EGA Buenos Aires, Argentina Prague Psychiatric Centre, Ústavní 91, 181 03 Prague 8, Czech Republic c Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Fleming Square 2, CZ166 10 Prague 6, Czech Republic b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 November 2009 Received in revised form 17 February 2010 Accepted 26 March 2010 Available online 3 April 2010
A procedure is described for the preparation of A-homo-5-pregnenes via an acid catalyzed rearrangement of cyclopropylcarbinols assisted by microwave irradiation. 3a-Hydroxy and 4a-hydroxy-A-homo-5pregnen-20-one, analogues of the neuroactive steroid allopregnanolone, were obtained by means of a regioselective epoxidation of a double bond in the expanded A-ring, using a fructose-derived chiral ketone as catalyst and oxone as oxidant. Although both these compounds were marginally active in inhibiting TBPS binding to GABAA receptors, 3b-hydroxy-A-homo-5-pregnen-20-one was almost as active as allopregnanolone. Reduction of the double bond of the latter compound resulted in a ten fold loss of activity. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: A-homopregnane Neurosteroid g-Aminobutyric acid GABAA receptor
1. Introduction The term “neuroactive steroid” (NAS) refers to steroids which, independent of their origin, are capable of modifying neural activities. It is now demonstrated that these neuroactive steroids positively or negatively modulate the function of members of the ligand-gated ion channel receptor superfamily [1e4]. Most of these studies have focused on their positive allosteric actions on g-amino butyric acid type A receptor (GABAA receptor) as those elicited by the endogenous steroids allopregnanolone (1) and pregnanolone (2). The physiological and pharmacological actions of neuroactive steroids are topics of widespread interest, since they have shown to be potent anticonvulsants, anxiolytics, and antistress agents as well as to possess sedative, hypnotic, and anesthetic activities. Structure-activity relationship studies of neuroactive steroids at GABAA receptors [4] have established a pharmacophore for positive modulation of the receptor by steroids, consisting of a hydrogen bond accepting group (such as COCH3 or CN) in a pseudoequatorial configuration at the 17b position and a hydrogen bond donating hydroxyl group in the 3a configuration.
* Corresponding author. Fax: þ54 11 45763385. E-mail address: [email protected] (G. Burton). 0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2010.03.037
The importance of the steric constraint imposed on the 3ahydroxyl group by the steroid A ring for GABAergic activities was examined by Covey and coworkers, who prepared a series of nonsteroidal analogues of 1 and 2 that mimicked parts of the steroid nucleus [5,6]. Among these analogues, perhydro benz[e] indenes as 3 and 4 were potent modulators of GABAA receptor function with certain analogues of 4 (with a modified side chain) displaying both potentiating and inhibitory actions [7]. Perhydro benz[e]indenes are steroidelike molecules in which the A-ring has been replaced by an open chain of appropriate length, giving the molecule considerable flexibility at the position originally occupied by the critical 3a hydrogen bond donor. The greater flexibility of benz[e]indenes would allow the 3-hydroxy group to mimic steroids having either a 3a or a 3b hydroxyl, thus being able to bind to the potentiating and the inhibitory sites on the GABAA receptor [3]. Consequently, those studies demonstrated that GABAergic activity does not require the 3a-hydroxyl group to be kept in a fixed position by a rigid A ring. To further explore these effects, we envisaged that a more controlled conformational mobility of the allopregnanolone A ring could be obtained by its expansion to a seven membered ring. Also the additional carbon in the resulting A-homopregnanes would allow further variations in the position of the A ring hydroxyl (e.g. 5 and 6).
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M.V. Dansey et al. / European Journal of Medicinal Chemistry 45 (2010) 3063e3069 Table 1 Formation of the A-homo steroid 8 from the cyclopropyl alcohol 7 via cationic rearrangement. Entry 1 2 3 4 5
Catalyst BF3-éter/CH2Cl2 Br2Zn/THF AlCl3/THF Br2Zn/THF AlCl3/THF
Conditions
0 C, 10 min 25 C, 28 hs 25 C, 3.5 hs MW 120 C, 10 min MW 65 C, 10 min
Yield (%)a 70b NDc NDc 87 80
ND: not determined. MW: microwave irradiation. a After chromatographic purification. b Data taken from ref. [8]. c Complex mixture.
2. Results and discussion 2.1. Chemistry In a previous publication we showed that treatment of cyclopropyl alcohol 7 with BF3-Et2O in dichloromethane gave A-homopregnadiene 8 in moderate yield (Scheme 1; Table 1, entry 1) [8]. Further attempts to improve the yield of this rearrangement using either zinc bromide or aluminum chloride as catalysts at room temperature were unsuccesful as the reaction was slow and gave complex mixtures of byproducts (Table 1, entries 3 and 4). However, when these reactions were carried out under microwave irradiation (MW) very good to excellent yields of the homosteroid (8) were obtained (entries 5 and 6). Treatment of the latter compound with LiAlH4 in THF gave alcohol 9. With the A-homosteroid 9 in hand, we focused our attention on the regio and stereoselective functionalization of the D3double bond. Epoxidation with m-chloroperbenzoic acid (MCPBA) gave a mixture of the undesired 5,6-epoxide and the 3,4:5,6diepoxide. Since this reaction was electronically favored at the most substituted D5-olefin, we turned to dioxirane mediated epoxidation, a highly efficient and stereospecific method towards both electron-rich and electron-deficient olefins [9,10]. Taking into account that the D5-olefin was sterically hindered, we used the chiral and bulky fructose-derived ketone 10 as catalyst and potassium monopersulfate (Oxone) as oxidant. Under these conditions, the 3b,4b-epoxide 11 was obtained as the only product in 56% yield after recovery of unreacted material (Scheme 2). The stereospecific b-epoxidation of diene 9 may be explained by the bent conformation of the ring A towards the a face in this compound [8]. Confirmation of the stereochemistry of epoxide 11 came from NMR data and molecular modelling of the A ring alcohols obtained by reductive cleavage. Treatment of epoxide 11 with LiAlH4 gave a 9:2 mixture of 3b-hydroxy (12) and 4b-hydroxy (13) A-homopregnenes (77% yield) that could be separated by flash chromatography. The 1H NMR spectrum of diol 13 showed the resonance of H-4 at d 3.54 as a triplet of triplets (J ¼ 10.2 and 5.0 Hz) indicating an axial orientation of this
Scheme 1. Reagents and conditions: (a) see Table 1. (b) i. LiAlH4, THF ii. 1 N HCl.
hydrogen. A strong correlation between H-4 and H-6 was observed in the NOESY spectrum; molecular modelling of all conformers of the seven membered A-ring in 13 showed that this was only possible in the most stable conformer of the 4b-alcohol (Fig. 1a). The b orientation of the 4-hydroxyl confirmed the b stereochemistry of epoxide 11 and hence of the hydroxyl at C-3 in 12. The 1H NMR spectrum of alcohol 12 showed an unresolved multiplet at d 4.05 (W1/2 ¼ 10.8 Hz) for H-3, typical of an equatorial hydrogen. To convert the 3b-oriented axial alcohol in 12 into the neurosteroid analogue 6, we first attempted an oxidation reduction sequence (Scheme 2). Thus diol 12 was oxidized with pyridinium
Scheme 2. Reagents and conditions: (a) ketone 10 (30% mol), Oxone, tetrabutylammonium acetate, K2CO3(aq), CH3CN/DME (1:2); (b) i. LiAlH4, THF ii. 1 M HCl; (c) PCC, BaCO3, MS 4Å, CH2Cl2; (d) 1 M K-Selectride, THF, 50 C.
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Fig. 2. Most stable conformer of compound 18 from HF/6-31G(d,p) calculations, showing NOEs and equatorial/axial orientation of relevant hydrogens. Other hydrogens are not shown for clarity.
Fig. 1. Most stable conformers from HF/6-31G(d,p) calculations, of compounds 13 (a), 15 (b), 5 (c) and 6 (d), showing NOEs and equatorial/axial orientation of relevant hydrogens. Other hydrogens are not shown for clarity. O(3)eC(20) distances for 3ahydroxy steroids are 5: 9.99 Å; 6: 10.72 Å.
chlorochromate to the diketone 14 followed by regioselective reduction of the ring A ketone with K-selectride (THF, 50 C). However, the resulting alcohol (15) had the 1H and 13C resonances of the A-ring atoms identical to those of 12, in particular it showed H-3 as an unresolved multiplet at d 4.05 (W1/2 ¼ 10.6 Hz)
Scheme 3. Reagents and conditions: (a) PPh3, HCOOH, DIAD, THF; (b) i. PCC, BaCO3, MS 4Å, CH2Cl2; ii. HCl, methanol, CH2Cl2.
indicating an axial orientation of the 3-hydroxyl. The NOESY spectrum of 15 had strong correlations among hydrogens at d 2.28 (4ab-H), 1.79 (1b-H), and 0.92 (19-H) and between the 4aa-H (d 1.90) and 6-H (d 5.41). Hydrogens at positions 4ab and 1b were assigned as axially oriented from their large vicinal coupling constants with 4a-H (J ¼ 13.7 Hz) and 2a-H (J ¼ 10.9 Hz) respectively, thus the b-orientation of the axial substituent at C-3 was confirmed. Molecular modelling of all conformers of the seven membered A-ring in 15 showed that, in the most stable conformer, the observed NOEs and couplings were consistent with the b orientation for the 3-hydroxyl (Fig. 1b) in agreement with the proposed configuration of diol 12. When the above sequence was applied to diol 13, the regioselective reduction of the intermediate diketone 16 occurred from the b face of ring A, giving the 4a-hydroxy analogue 5 as the only product in 70% yield (Scheme 2). The inversion of the stereochemistry at C-4 was evident from the change in the H-4 resonance (compared to 13), observed as an unresolved multiplet at d 3.94 (W1/2 ¼ 17.3 Hz) indicative of an equatorial hydrogen. The NOESY spectrum of 5 did not show a correlation between H-4 and H-6, but showed strong correlations among hydrogens at d 1.05 (1b), d 2.46 (4ab) and d 0.94 (H-19) which were indicative of the axial orientation of H-1b and H-4ab. The NOE correlation of both these hydrogens with H-3b (d 1.36) confirmed also its b-axial position, and the a orientation of the axial hydroxyl at C-4 (Fig. 1c). In view of the above results, the conversion of diol 12 into the 3a alcohol 6 was carried out by inversion of the alcohol at C-3 using the Mitsunobu reaction (Scheme 3) [11,12]. Thus, treatment of 12 with DIAD/Ph3P/HCO2H gave the 3a-formyloxy pregnane 17. Oxidation of 17 with pyridinium chlorochromate, followed by mild hydrolysis with potassium carbonate in methanol gave the target compound 6 (40% yield from 12). The inversion of stereochemistry at C-3 was evident from the change in the H-3 resonance (compared to 12 and 15) observed as a triple double doublet, with large axial-axial couplings with H-2a and H-4a (J ¼ 10.5 Hz), and smaller equatorial-axial couplings with H-2b and H-4b (J ¼ 3.7 and 5.5 Hz). The NOESY spectrum of 6 showed strong correlations of H-3 with H-1b (d 1.18), H-2b (1.64), H-4b (d 2.19) and H-4ab (1.99). The latter hydrogen and H-1b also had NOE correlations with H-19 (d 0.88) confirming the a orientation of the hydroxyl at C-3 (Fig. 1d). Hydrogenation of compound 15 gave the reduced 5a-pregnane 18 with good stereoselectivity (5a/5b 9:1 determined by 1H NMR of the crude reduction product). The stereochemistry at position 5 in compound 18 was inferred from the 13C NMR data. Thus, the C-19 resonance at d 13.8 for the 5a isomer was especially indicative of an A/B trans-fused steroid in comparison with the C-19 resonance at d 21.5 for the 5b isomer (observed as a minor component in the 13C NMR spectrum of the crude reduction product).[13] The NOESY spectrum of 18 showed correlations between H-5 (d 0.73) and hydrogens at positions 1a (d 1.10), 7a (d 0.88) and 9a (d 1.07). No
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correlation was observed between H-19 and H-5, confirming the a orientation of the hydrogen at C-5. The H-3 resonance in 18 appeared at d 3.94 as a double double triplet (J ¼ 8.8, 3.6 and 5.7 Hz) in contrast with the unresolved multiplet observed for compound 15, indicating a major change in the conformation of ring A. Fig. 2 shows the most stable conformer of 18 (from HF/6-31G(d,p) calculations), and the observed NOEs in good agreement with the proposed structure.
Fig. 3. Superposition of conformer 2 of A-homopregnane 15 (blue) and pregnanolone (2) (white) from HF/6-31G(d,p) calculations. Confomer 2 is 3.30 kcal/mol less stable than the most stable conformer of 15. Overlay corresponds to best fit for carbons 20, 16, 13, 11, 10 and 8 and O(3) (RMS error 0,22 Å); O(3)eC(20) distances are 2: 9.61 Å; 15: 9.93 Å.
3. Conclusion
2.2. Receptor binding studies GABAA receptor activity was evaluated by assaying the effect of the synthetic analogues 5, 6, 15 and 18 on the binding of [35S]-tertbutyl-bicyclo[2.2.2]phosphorothionate (TBPS). The binding of this convulsant in the presence of GABA closely reflects the functional state of GABAA receptors and may be useful for characterization of allosteric interactions between various sites on the receptor [4,14]. Allopregnanolone (1) was used as a positive control to check the viability of the methods. As shown in Table 2, compounds 5 and 6 with an a-oriented hydroxyl in ring A had a IC50 in the micromolar range; ab initio calculations show that both compounds adopt an overall bent conformation that resembles that of pregnanolone (2) (Fig. 1c and d), however attempts to overlay the structures of 5 and 6 with 2 resulted in a poor overall fit. Interestingly the 3b-alcohol 15 displayed an IC50 similar to that of allopregnanolone (1) but with a lower maximal inhibition. Although the most stable conformer of 15 has the 3-hydroxyl in an opposite orientation compared to allopregnanolone (1) and pregnanolone (2) (Fig. 1b), ab initio calculations showed two other conformers within 3.43 kcal/mol of the most stable one. One of these conformers, had an excellent fit with pregnanolone (Fig. 3), the 3-hydroxyl and the C-17 side chain occupying positions close in space for both compounds. Further support to this observation came from reduction of the 5,6 double bond in 15 to give 3b-alcohol 18. In this compound, the presence of the 5a-H and a sp3 carbon at position 5 reduces the flexibility of the A ring and precludes compound 18 from adopting a conformation smilar to that of pregnanolone. When compound 18 was assayed for its effect on TBPS binding, a ten fold loss of activity was observed.
Table 2 Inhibition of binding of [35S]-tert-butylbicyclo[2.2.2]phosporothionate ([35S]TBPS) to membranes from rat cerebellum by A-homo steroids 5, 6, 15 and 18. Compound
Imax (%)a
IC50 (nM)b
4a-hydroxy-A-homo-5-pregnen-20-one (5) 3a-hydroxy-A-homo-5-pregnen-20-one (6) 3b-hydroxy-A-homo-5-pregnen-20-one (15) 3b-hydroxy-A-homo-5aH-pregnan-20-one (18) Allopregnanolone (1)c
50.7 13.7 75.6 17.3 50.0 10.6 46.2 15.4 79.6 4.8
800 21.8 1200 553 100 55 1000 20 80 23
a b c
The maximal suppression of the binding. The steroid concentration producing a half-maximal inhibition. Data taken from ref. [17].
A series of four A-homo steroids related to the neurosteroids pregnanolone and allopreganolone have been synthesized and their GABAA receptor activity was evaluated in vitro. The conformational mobility of the seven-membered A-ring combined with the enhanced flexibility at the A/B ring junction introduced by the 5,6 double bond in ring B, would allow the 3b-hydroxy steroid 15 to mimic the pregnanolone molecule with a minor energy penalty, thus attaining an activity comparable to the natural neuroactive steroids.
4. Experimental 4.1. General Mps were taken on a Fisher-Johns apparatus and are uncorrected. IR spectra were recorded in thin films using KBr disks on a Nicolet Magna 550 FT-IR spectrophotometer, values are given in cm1. 1H and 13C NMR spectra were recorded on a Bruker Avance II 500 at 500.13 and 125.77 MHz respectively, in deuterochloroform. Chemical shifts are given in ppm downfield from TMS as internal standard, J values are given in Hz. Multiplicity determinations and 2D spectra (COSY, HSQC and HMBC) were obtained using standard Bruker software. The electron impact mass spectra (MS) were collected on a Shimadzu QP-5000 mass spectrometer at 70 eV by direct inlet. High resolution mass spectra (HRMS) were measured on an Agilent LCTOF, high resolution TOF analyzer or a Bruker micrOTOF-Q II spectrometer with ESI ionization. Elemental analysis was performed on an EAI Exeter Analytical, Inc. CE-440 apparatus. Microwave assisted reactions were carried out on a CEM Discover reactor, mode Discover (closed vessel, Power max: on). Vacuum liquid chromatography (VLC) and column flash chromatography were carried out on silica gel 60-G (Merck) and silica gel S 0.040e0.063 mm respectively. Thin layer chromatography (tlc) analysis was performed on silica gel 60 F254 (0.2 mm thick). The homogeneity of all compounds was confirmed by thin layer chromatography. Solvents were evaporated at reduced pressure and ca. 40e50 C. 3b-Hydroxy-20b-acetyloxy-4b,5b-methylenepregnane (7) was obtained from progesterone following the procedure previously described by us [8]. The ketone catalyst 10 was prepared from fructose, following the procedure described by Yian Shi et al. [15]. Geometry optimizations were carried out with the ab-initio quantum chemistry program GAUSSIAN 03 and the HF/6-31G(d,p) basis set [16].
M.V. Dansey et al. / European Journal of Medicinal Chemistry 45 (2010) 3063e3069
4.2. Chemical synthesis 4.2.1. A-homopregna-3,5-dien-20b-yl acetate (8) Method A: 3b-Hydroxy-4b,5b-methylenepregnan-20b-yl acetate (7) (0.030 g, 0.08 mmol) was dissolved in dry tetrahydrofuran (3 mL) under argon atmosphere. AlCl3 (0.063 g, 0.47 mmol) was added and the mixture was stirred in a microwave reactor at 65 C, 100 psi and 300 W for 10 min. The reaction mixture was diluted with aqueous NaHCO3 (5%) and extracted with dichloromethane. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification by flash chromatography (hexane-ethyl acetate, 97.5:2.5) gave diene 8 (0.023 g, 80%) as an amorphous solid identical (tlc, NMR) to that described previously [8]. Method B: 3b-Hydroxy-4b,5b-methylenepregnan-20b-yl acetate (7) (0.030 g, 0.08 mmol) was dissolved in dry tetrahydrofuran (3 mL) under argon atmosphere. ZnBr2 (0.120 g, 0.53 mmol) was added and the mixture was stirred in a microwave reactor at 120 C, 100 psi and 300 W for 10 minutes. Extractive work-up as above, gave diene 8 (0.025 g, 87%) as an amorphous solid identical (tlc, NMR) to that described previously [8]. 4.2.2. 20b-Hydroxy-A-homopregna-3,5-diene (9) To a solution of acetate 8 (0.120 g, 0.337 mmol) in diethyl ether (5 mL) under nitrogen atmosphere was added LiAlH4 (0.038 g, 0.99 mmol). After stirring for 1 h at room temperature HCl (1 N, 1 mL) was added, the mixture was poured onto an aqueous saturated solution of sodium and potassium tartrate and extracted with dichloromethane. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification by flash chromatography gave alcohol 9 (0.078 g, 74%) as a white crystalline solid identical (tlc, NMR) to that described previously [8]. 4.2.3. 3b,4b-Epoxy-20b-hydroxy-A-homo-5-pregnene (11) To a solution of compound 9 (0.250 g, 0.795 mmol) in acetonitrileedimethoxyethane (10.5 mL, 1:2, v/v) were succesively added ketone 10 (0.065 g, 0.252 mmol), tetrabutylammonium acetate (0.0046 mg, 0.028 mmol) and Na2(EDTA) (0.0013 g, 0.004 mmol). A solution of Oxone (1.312 g, 2.1 mmol) in aqueous Na2(EDTA) (4 104 M, 3.7 mL) and a solution of K2CO3 (0.987 g, 7.15 mmol) in water (3.7 mL), were then added simultaneously to the stirred mixture. Another 3.7 mL of each solution was added using a dual syringe pump (4 mL/h). Stirring was continued at room temperature for 30 min, the reaction was poured into HCl (1 N, 7.15 mL), and extracted with dichloromethane. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification by flash chromatography (hexane-ethyl acetate, 9:1) gave unreacted 9 (0.100 g, 40%) and epoxide 11 (0.088 g, 35%) as a white solid; mp 179e181 C (from hexaneeethyl acetate); Anal. calcd. for C22H34O2: C 79.95 H 10.4 found C 79.6 H 10.7; nmax (KBr, cm1) 3413, 2941, 2868, 1117, 972 and 881; 1H NMR dH: 5.38 (1 H, dd, J ¼ 4.2 and 1.2 Hz, H-6), 3.74 (1 H, m, H-20), 3.14 (1 H, m, H-4), 2.96 (1 H, m, H-3), 2.48 (1 H, dd, J ¼ 14.1 and 8.2 Hz, H-4aa), 2.36 (1 H, br d, J ¼ 14.1 Hz, H-4ab), 2.08 (1 H, dt, J ¼ 12.5 and 3.4 Hz, H-12b), 2.00 (1 H, m, H-7b), 1.95 (1 H, m, H-2a), 1.85 (1 H, m, H-2b), 1.67 (1 H, m, H-16b), 1.65 (1 H, m, H-15a), 1.57 (1 H, m, H-7a), 1.56 (1 H, m, H-8), 1.52 (1 H, m, H-11a), 1.41 (1 H, m, H-11b), 1.35 (3 H, m, H-1 and H-17), 1.27 (1 H, m, H-12a), 1.20 (1 H, m, H16a), 1.15 (3 H, d, J ¼ 6.0 Hz, H-21), 1.14 (1 H, m, H-15b), 1.14 (1 H, m, H-9), 1.08 (1 H, m, H-14), 0.87 (3 H, s, H-19), 0.76 (3 H, s, H-18); 13C NMR dC: 141.0 (C-5), 123.8 (C-6), 70.6 (C-20), 58.5 (C-17), 56.5 (C-14 and C-9), 55.6 (C-4), 55.3 (C-3), 42.2 (C-13), 40.3 (C-10), 40.0 (C12), 32.6 (C-4a), 31.6 (C-8 and C-7), 25.8 (C-1), 25.7 (C-16), 24.5 (C15), 23.7 (C-21), 23.2 (C-19), 23.0 (C-2), 21.3 (C-11), 12.5 (C-18); MS
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(EI) m/z (%): 330 (Mþ, 3), 315 (2), 207 (5), 189 (11), 163 (9), 91 (18), 55 (24), 45 (100). 4.2.4. 3b,20b-dihydroxy-A-homo-5-pregnene (12) and 4b,20bdihydroxy-A-homo-5-pregnene (13) To a stirred solution of epoxide 11 (0.330 g, 1 mmol) in dry tetrahydrofuran (33.5 mL) was added LiAlH4 (0.015 g, 1.6 mmol) under nitrogen atmosphere. After stirring for 20 min at room temperature, the reaction mixture was neutralized with 1 N HCl, poured onto an aqueous saturated solution of sodium and potassium tartrate and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification by flash chromatography (hexane-ethyl acetate) gave alcohols 12 (0.212 g, 63%) and 13 (0.047 g, 14%). Compound 12, amorphous solid; Anal. calcd. for C22H36O2: C 79.5 H 10.9 found C 79.7 H 10.8; nmax (KBr, cm1) 3388, 2954, 1486, 1052 and 749; 1H NMR dH: 5.41 (1 H, dd, J ¼ 5.8 and 2.2 Hz, H-6), 4.05 (1 H, bs, W1/2 ¼ 10.8 Hz, H-3), 3.75 (1 H, m, H-20), 2.30 (1 H, m, H-4ab), 2.09 (1 H, dt, J ¼ 12.6 and 3.4 Hz, H-12b), 2.02 (1H, dd, J ¼ 12.1 and 5.2 Hz, H-7b), 1.93 (1 H, m, H-4b), 1.92 (1 H, m, H-4aa), 1.76 (1 H, dd, J ¼ 15.0 and 11.2 Hz, H-1b), 1.69 (1 H, m, H-16b), 1.65 (1 H, m, H-15a), 1.63 (1 H, m, H-2b), 1.60 (1 H, m, H-11a), 1.59 (1 H, m, H-8), 1.58 (1 H, m, H-7a), 1.53 (1 H, m, H-1a), 1.50 (1 H, m, H-12a), 1.49 (1 H, m, H-4a), 1.46 (1 H, m, H-2a), 1.45 (1 H, m, H-11b), 1.35 (2 H, m, H-17), 1.22 (1 H, m, H-15b), 1.20 (1 H, m, H-16a), 1.16 (1 H, m, H-9), 1.15 (3 H, d, J ¼ 6.2 Hz, H-21), 1.08 (1 H, m, H-14), 0.93 (3 H, m, H-19), 0.79 (3 H, m, H-18); 13C NMR dC: 146.9 (C-5), 122.6 (C-6), 70.6 (C-20), 68.0 (C-3), 58.5 (C-17), 56.7 (C-14), 44.1 (C-9), 42.3 (C-13), 40.3 (C-4), 40.1 (C-12), 40.05 (C-10), 31.8 (C-8), 31.6 (C-7), 29.5 (C1), 26.9 (C-4a), 26.6 (C-2), 25.8 (C-16), 24.5 (C-15), 23.8 (C-19), 23.6 (C-21), 21.5 (C-11), 12.5 (C-18); MS (EI) m/z (%): 332 (Mþ, 19), 314 (10), 286 (23), 233 (46), 189 (100), 163 (45), 121 (48) 95 (48); HRMS (ESI) m/z: 333.2790 (Mþ þ H, C22H37O2 requires 333.2788). Compound 13 white solid; mp 179e180 C (from hexane-ethyl acetate); nmax (KBr, cm1) 3263, 2927, 1269, 1098, 1038, 1019 and 808; 1H NMR dH 5.52 (1 H, dd, J ¼ 5.5 and 1.8 Hz, H-6), 3.74 (1 H, dq, J ¼ 9.8 and 6.1 Hz, H-20), 3.54 (1 H, tt, J ¼ 10.2 and 5.0 Hz, H-4), 2.24 (1 H, dd, J ¼ 13.0 and 5.0 Hz, H4aa), 2.20 (1H, br t, J ¼ 12.4 Hz, H4ab), 2.07 (2 H, m, H-3a), 2.06 (1H, dt, J ¼ 12.5 and 3.5 Hz, H-12b), 2.03 (1H, m, H-7b), 1.94 (1 H, dd, J ¼ 14.5 and 8.8 Hz, H-1a), 1.67 (1 H, m, H16b),1.63 (1 H, m, H-15a),1.55 (2 H, m, H-8 and H-7a),1.54 (1 H, m, H11a), 1.43 (1 H, m, H-2a), 1.41 (1 H, m, H-11b), 1.33 (1 H, br q, J ¼ 9.8 Hz, H-17),1.25 (1 H, m, H-12a),1.21 (1 H, m, H-3b),1.17 (1 H, m, H-16a),1.16 (1 H, m, H-1b),1.14 (1 H, m, H-15b),1.14 (3 H, d, J ¼ 6.1 Hz, H-21), 1.08 (2 H, m, H-9 and H-2b), 1.06 (1 H, m, H-14), 0.91 (3 H, s, H19), 0.78 (3 H, s, H-18); 13C NMR dC: 140.7 (C-5), 125.4 (C-6), 76.1 (C4), 70.6 (C-20), 58.5 (C-17), 56.6 (C-14), 44.0 (C-9), 42.3 (C-13), 42.0 (C-4a), 40.4 (C-3), 40.1 (C-12), 40.0 (C-10), 35.9 (C-1), 31.6 (C-8), 31.5 (C-7), 25.7 (C-16), 24.4 (C-15), 23.7 (C-19), 23.6 (C-21), 21.4 (C-11), 18.8 (C-2), 12.5 (C-18); MS (EI) m/z (%): 332 (Mþ, 29), 314 (24), 233 (65), 189 (100), 163 (51), 119 (69), 105 (74), 55(83), 45 (91); HRMS (ESI) m/z: 333.2784 (Mþ þ H, C22H37O2 requires 333.2788). 4.2.5. A-homo-5-pregnene-3,20-dione (14) A solution of alcohol 12 (0.059 g, 0.18 mmol) in dichloromethane (19 mL) was added to a stirred suspension of PCC (0.364 g, 1.65 mmol), BaCO3 (0.101 g, 0.52 mmol) and activated 4Å molecular sieves (0.144 g) in dry dichloromethane (10 mL). After stirring at room temperature for 4 h, celite (1 g) was added and the solvent was removed under reduced pressure. The mixture was percholated through a short silica gel column (hexaneeethyl acetate) and the residue obtained after evaporation of the solvent was purified by flash chromatography (hexaneeethyl acetate, 95:5) to give diketone 14 (0.039 g, 66%) as a white solid; mp 166-167 C (from hexane-ethyl acetate); nmax (KBr, cm1) 2934, 1703, 1354 and 1236;
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H NMR dH 5.57 (1 H, dd, J ¼ 5.2 and 2.0 Hz, H-6), 2.63 (1 H, dt, J ¼ 14.7 and 4.7 Hz, H-4b), 2.56 (1 H, t, J ¼ 8.7 Hz, C-17), 2.45 (1 H, m, H-4ab), 2.36 (1 H, m, H-4a), 2.32 (2 H, m, H-2), 2.19 (1 H, m, H-16b), 2.16 (1 H, m, H-4aa), 2.13 (3 H, s, H-21), 2.08 (1 H, m, H-12b), 2.06 (1 H, m, H-7b), 1.76 (2 H, m, H-1), 1.70 (1 H, m, H-15a), 1.69 (1 H, m, H16a), 1.63 (1 H, m, H-11a), 1.61 (1 H, m, H-7a), 1.58 (1 H, m, H-8), 1.48 (1 H, m, H-12a), 1.47 (1 H, m, H-11b), 1.23 (1 H, m, H-14), 1.22 (1 H, m, H-9), 1.21 (1 H, m, H-15b), 1.00 (3 H, s, H-19), 0.65 (3 H, s, H-18); 13 C NMR dC: 213.1 (C-3), 209.4 (C-20), 142.7 (C-5), 125.2 (C-6), 63.6 (C-17), 57.1 (C-14), 44.6 (C-9), 44.1 (C-4), 43.9 (C-13), 38.9 (C-2 and C-10), 38.8 (C-12), 31.8 (C-8), 31.5 (C-21), 31.3 (C-7), 31.1 (C-1), 27.9 (C-4a), 24.4 (C-15), 22.9 (C-16), 22.0 (C-19), 21.5 (C-11), 13.3 (C-18); MS (EI) m/z (%): 328 (Mþ, 55), 310 (23), 300 (30), 205 (35), 119 (32), 105 (35), 55 (39), 43 (100); HRMS (ESI) m/z: 329.2476 (Mþ þ H, C22H33O2 requires 329.2475). 4.2.6. A-homo-5-pregnene-4,20-dione (16) Alcohol 13 (0.0086 g, 0.029 mmol) was oxidized with PCC (0.054 g, 0.245 mmol) following the procedure described for compound 12, to give diketone 16 (0.0064 g, 75%) as a white solid; mp 141-144 C (from hexane-ethyl acetate); nmax (KBr, cm1) 2918, 1701, 1541, 1508.1, 1456 and 1356; 1H NMR dH: 5.57 (1 H, dd, J ¼ 5.5 and 2.1 Hz, H-6), 3.26 (1 H, d, J ¼ 14.4 Hz, H-4aa), 2.84 (1 H, d, J ¼ 14.4 Hz, H-4ab), 2.62 (1 H, m, H-3a), 2.55 (1 H, t, J ¼ 9.1 Hz, H17), 2.22 (1 H, m, H-3b), 2.19 (1 H, m, H-16b), 2.13 (3 H, s, H-21), 2.09 (1 H, m, H-7b), 2.08 (1 H, m, H-12b), 1.87 (1 H, dd, J ¼ 13.0 and 7.1 Hz, H-1a), 1.68 (1 H, m, H-16a), 1.67 (1 H, m, H-15a), 1.66 (1 H, m, H-11a), 1.65 (1 H, m, H-2a), 1.63 (1 H, m, H-7a), 1.60 (1 H, m, H-8), 1.57 (2 H, m, H-2b and H-11b), 1.55 (1 H, m, H-1b), 1.48 (1 H, m, H12a), 1.30 (1 H, m, H-9), 1.25 (1 H, m, H-15b), 1.23 (1 H, m, H-14), 0.99 (3 H, s, H-19), 0.65 (3 H, s, H-18); 13C NMR dC: 210.7 (C-4), 209.5 (C-20), 137.2 (C-5), 127.1 (C-6), 63.6 (C-17), 57.1 (C-14), 48.6 (C-4a), 44.0 (C-9), 43.9 (C-13), 42.5 (C-3), 39.8 (C-10), 38.8 (C-12), 34.9 (C1), 31.7 (C-8), 31.6 (C-7), 31.5 (C-21), 24.4 (C-15), 22.9 (C-16), 22.7 (C-19), 21.4 (C-11), 18.6 (C-2), 13.3 (C-18); MS (EI) m/z (%): 328 (Mþ, 70), 205 (24), 187 (28), 105 (46), 85 (54), 55 (56), 43 (100); HRMS (ESI) m/z: 329.2475 (Mþ þ H, C22H33O2 requires 329.2475). 4.2.7. 3b-Hydroxy-A-homo-5-pregnen-20-one (15) A solution of K-Selectride in tetrahydrofuran (1 M, 0.22 mL, 0.22 mmol) was added to a stirred solution of diketone 14 (0.060 g, 0.183 mmol) in dry tetrahydrofuran (5 mL) at -50 C, under nitrogen atmosphere. After 30 min the reaction mixture was poured onto an aqueous ammonium chloride solution (5%) and extracted with dichloromethane. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification by flash chromatography (hexane-ethyl acetate, 85:15) gave the 3b-alcohol 15 (0.038 g, 63%) as a white solid; mp 151 153 C (from hexaneethyl acetate); Anal. calcd. for C22H34O2: C 79.95 H 10.4 found: C 79.9 H 10.4; nmax (KBr, cm1) 3329, 2934, 1710 and 1038; 1H NMR dH: 5.41 (1 H, dd, J ¼ 5.1 and 1.6 Hz, H-6), 4.05 (1 H, br s, W1/2 ¼ 10.6 Hz, H-3), 2.53 (1 H, t, J ¼ 9.0 Hz, H-17), 2.28 (1 H, dt, J ¼ 3.1 and 13.7 Hz, H-4ab), 2.19 (1 H, m, H-16b), 2.13 (3 H, s, H-21), 2.06 (1 H, m, H-12b), 2.05 (1 H, m, H-7b), 1.92 (1 H, m, H-4b), 1.90 (1 H, m, H-4aa), 1.79 (1 H, dd, J ¼ 15.2 and 10.9 Hz, H-1b), 1.68 (2 H, m, H-15a and H-11a), 1.67 (1 H, m, H-16a), 1.63 (1 H, m, H-2b), 1.57 (1 H, m, H-7a), 1.56 (1 H, m, H-8), 1.51 (1 H, dd, J ¼ 15.2 and 8.3 Hz, H-1a), 1.48 (1 H, m, H-4a), 1.46 (1 H, m, H-12a), 1.44 (1 H, m, H-11b), 1.43 (1 H, m, H-2a), 1.23 (1 H, m, H15b), 1.20 (1 H, m, H-14), 1.16 (1 H, m, H-9), 0.92 (3 H, s, H-19), 0.64 (3 H, s, H-18); 13C NMR dC: 209.6 (C-20), 146.8 (C-5), 122.4 (C-6), 67.8 (C3), 63.7 (C-17), 57.4 (C-14), 44.02 (C-13), 44.00 (C-9), 40.3 (C-4), 40.0 (C-10), 39.0 (C-12), 31.9 (C-8), 31.5 (C-21), 31.3 (C-7), 29.4 (C-1), 26.9 (C-4a), 26.6 (C-2), 24.3 (C-15), 23.7 (C-19), 22.8 (C-16), 21.6 (C-11), 13.8 (C-18); MS (EI) m/z (%): 330 (Mþ, 23), 312 (15), 302 (14), 231 (46), 205 (22), 187 (25), 121 (36), 43 (100).
4.2.8. 4a-Hydroxy-A-homo-5-pregnen-20-one (5) Diketone 16 (0.007 g, 0.021 mmol) was reduced with 1 M Kselectride in dry tetrahydrofuran (0.025 mL, 0.025 mmol) following the procedure described for compound 14, to give 5 (0.005 g, 70%) as an amorphous solid; Anal. calcd. for C22H34O2: C 79.95 H 10.4 found C 79.9 H 10.4; nmax (KBr, cm1) 2928, 1705, 1541 and 1458; 1H NMR dH: 5.52 (1 H, dd, J ¼ 4.9 and 1.9 Hz, H-6), 3.94 (1 H, br s, W1/2 ¼ 15.0 Hz, H-4), 2.54 (1 H, t, J ¼ 9.0 Hz, H-17), 2.46 (1 H, br d, J ¼ 13.6 Hz, H-4ab), 2.22 (1 H, dd, J ¼ 13.6 and 1.0 Hz, H-4aa), 2.20 (1 H, m, H-16b), 2.13 (3 H, s, H-21), 2.11 (1 H, dt, J ¼ 16.8 and 4.7 Hz, H-7b), 2.06 (1 H, dt, J ¼ 12.0 and 3.1 Hz, H-12b), 1.98 (1 H, dd, J ¼ 14.4 and 8.9 Hz, H-1a), 1.87 (1 H, br d, J ¼ 14.3 Hz, H-3a), 1.70 (2 H, m, H-15a and H-16a), 1.69 (2 H, m, H-11a and H-7a), 1.64 (1 H, m, H-8), 1.48 (1 H, dt, J ¼ 2.9 and 12.0 Hz, H-12a), 1.41 (1 H, m, H-11b), 1.36 (1 H, m, H-3b), 1.33 (1 H, m, H-2b), 1.25 (1 H, m, H-15b), 1.23 (1 H, m, H-14),1.20 (1 H, m, H-9), 1.12 (1 H, m, H-2a),1.05 (1 H, dd, J ¼ 14.4 and 10.0 Hz, H-1b), 0.94 (3 H, s, H19), 0.64 (3 H, s, H-18); 13C NMR dH: 209.6 (C-20),140.1 (C-5),126.7 (C6), 66.0 (C-4), 63.6 (C-17), 57.3 (C-14), 44.5 (C-10), 44.02 (C-13), 44.00 (C-9), 39.3 (C-3), 39.0 (C-12), 38.3 (C-4a), 36.7 (C-1), 31.8 (C-8), 31.5 (C-21), 30.9 (C-7), 24.3 (C-15), 24.0 (C-19), 22.9 (C-16), 21.6 (C-11),17.1 (C-2), 13.4 (C-18); MS (EI) m/z (%): 330 (Mþ, 1), 312 (5), 149 (23), 105 (21), 85 (27), 55 (19), 43 (100). 4.2.9. 3a-Hydroxy-A-homo-5-pregnen-20-one (6) A solution of diisopropyl azodicarboxylate (0.027 mL, 0.12 mmol) in dry tetrahydrofuran (0.116 mL) was added to a solution of diol 12 (0.020 g, 0.060 mmol), triphenylphosphine (0.032 g, 0.12 mmol) and formic acid (0.0048 mL, 0.12 mmol), in dry tetrahydrofuran (0.75 mL). After stirring for 20 h at room temperature, the reaction mixture was diluted with CH2Cl2 and silica gel (0.100 g) was added. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (hexane to hexaneeethyl acetate, 95:5), to give formate 17 (0.016 g, 71%) as an amorphous solid; 1H NMR dH: 5.49 (1 H, dd, J ¼ 4.8 and 1.6 Hz, H-6), 4.80 (1 H, m, H-3), 3.72 (1 H, m, H-20), 1.15 (3 H, d, J ¼ 6.2 Hz, H-21), 0.90 (3 H, s, H-19), 0.80 (3 H, s, H-18); 13C NMR dC: 70.6 (C-20), 145.4 (C-5), 123.9 (C-6), 37.3 (C-4), 58.5 (C-17), 56.6 (C-14), 44.2 (C-9), 42.2 (C-13), 27.7 (C-4a), 76.5 (C-3), 40.26 (C-10), 40.0 (C-12), 31.6 (C8), 31.5 (C-7), 30.8 (C-1), 28.7 (C-2), 25.7 (C-16), 24.4 (C-15), 23.6 (C21), 23.3 (C-19), 21.4 (C-11), 12.5 (C-18). A solution of formate 17 (0.015 g, 0.04 mmol) in dichloromethane (1.5 mL) was added to a stirred suspension of PCC 0.036 g, 0.165 mmol), BaCO3 (0.015 g, 0.080 mmol) and 4Å molecular sieves (0.030 g) in dry dichloromethane (1 mL). After stirring for 1 h at room temperature, celite (0.100 g) was added and the mixture was percholated through a silica gel column (hexaneeethyl acetate, 95:5), to give the 20ketone (0.009 g, 60%) as an amorphous solid; 1H NMR dH: 5.49 (1 H, dd, J ¼ 5.5 and 2.0 Hz, H-6), 4.84 (1 H, m, H-3), 2.12 (3 H, s, H-21), 0.89 (3 H, s, H-19), 0.63 (3 H, s, H-18); 13C NMR dC: 209.6 (C-20), 145.3 (C-5), 123.8 (C-6), 37.4 (C-4), 63.7 (C-17), 57.3 (C-14), 44.2 (C9), 44.0 (C-13), 27.8 (C-4a), 76.6 (C-3), 39.0 (C-12), 40.3 (C-10), 31.7 (C-8), 31.3 (C-7), 30.9 (C-1), 28.7 (C-2), 22.9 (C-16), 24.4 (C-15), 31.6 (C-21), 23.3 (C-19), 21.6 (C-11), 13.4 (C-18). To the 20-ketone obtained above (0.009 g, 0.02 mmol) in a mixture of dichloromethane (0.16 mL), methanol (0.53 mL) and water (0.04 mL), was added conc. HCl (0.078 mL, 0.94 mmol). After stirring for 1 h at room temperature, the reaction mixture was neutralized with aqueous NaHCO3 and extracted with dichloromethane. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification by preparative tlc (hexane-ethyl acetate, 7:3) gave compound 6 (0.008 g, 100%) as a white solid; mp 165e170 C (from hexane-ethyl acetate); Anal. calcd. for C22H34O2: C 79.95 H 10.4 found C 79.8 H 10.4; nmax (KBr, cm1) 3473, 2928, 1697, 14589, 1043; 1H NMR dH: 5.47 (1 H, dd, J ¼ 5.0 and 2.0 Hz, H-6), 3.60 (1 H, tdd, J ¼ 10.5, 5.5 and 3.7 Hz, H-3),
M.V. Dansey et al. / European Journal of Medicinal Chemistry 45 (2010) 3063e3069
2.54 (1 H, t, J ¼ 8.4 Hz, H-17), 2.19 (2 H, m, H-16b and H-4b), 2.12 (3 H, s, H-21), 2.05 (2 H, m, H-12b and H-7b), 1.99 (2 H, m, H-4aa and H-4ab), 1.82 (1 H, dd, J ¼ 15.1 and 9.5 Hz, H-1a), 1.69 (1 H, m, H-15a), 1.66 (2 H, m, H-11a and H-16a), 1.64 (1 H, m, H-2b), 1.59 (1 H, m, H7a), 1.55 (1 H, m, H-8), 1.47 (1 H, dt, J ¼ 2.8 and 12.8 Hz, H-12a), 1.41 (1 H, dq, J ¼ 3.2 and 12.8 Hz, H-11b), 1.31 (1 H, dt, J ¼ 13.3 and 10.5 Hz, H-2a), 1.23 (1 H, m, H-15b), 1.22 (1 H, m, H-4a), 1.21 (1 H, m, H-14), 1.18 (1 H, dd, J ¼ 15.1 and 10.5 Hz, H-1b), 1.17 (1 H, m, H-9), 0.88 (3 H, s, H-19), 0.63 (3 H, s, H-18); 13C NMR dC: 209.6 (C-20), 145.9 (C-5), 123.3 (C-6), 74.9 (C-3), 63.7 (C-17), 57.3 (C-14), 44.2 (C9), 44.0 (C-13), 41.4 (C-4), 40.4 (C-10), 39.0 (C-12), 32.5 (C-2), 31.7 (C-8), 31.5 (C-21), 31.3 (C-7), 31.1 (C-1), 28.0 (C-4a), 22.9 (C-16), 24.3 (C-15), 23.3 (C-19), 21.6 (C-11), 13.3 (C-18); MS (EI) m/z (%): 330 (Mþ, 16), 312 (34), 284 (41), 231 (20), 187 (21), 91 (45), 79 (45), 43 (100); HRMS (ESI) m/z: 331.2633 (Mþ þ H, C22H35O2 requires 331.2632), 313.2528 (Mþ þ HeH2O, C22H33O requires 313.2526). 4.2.10. 3b-Hydroxy-A-homo-5aH-pregnan-20-one (18) 10% Palladium on carbon (0.060 g) was added to a solution of steroid 15 (0.020 g, 0.061 mmol) in ethyl acetate (5 mL). The suspension was hydrogenated for 22 h at 60 psi and 25 C, the reaction mixture was filtered through a silica gel column and the solvent was evaporated under reduced pressure. The residue (a 9:1 mixture of 5aH and 5bH steroids as determined by 1H NMR) was purified by preparative tlc (hexane-ethyl acetate, 8:2) to give homopregnane 18 (0.016 g, 80%) as a white solid; mp 148-150 C (from hexane-ethyl acetate); Anal. calcd. for C22H36O2: C 79.5 H 10.9 found C 79.4 H,10.7; nmax (KBr, cm1) 3421, 2926, 1695, 1541, 1458 and 1038; 1H NMR dH: 3.94 (1 H, ddt, J ¼ 8.8, 3.6 and 5.7 Hz, H-3), 2.51 (1 H, t, J ¼ 9.0 Hz, H-17), 2.14 (1 H, m, H-16b), 2.11 (3 H, s, H-21), 2.00 (1 H, dt, J ¼ 12.4 and 3.3 Hz, H-12b), 1.91 (1 H, m, H-4a), 1.75 (1 H, m, H-1b), 1.73 (1 H, m, H-2a), 1.69 (2 H, m, H-4aa and H-11a), 1.65 (1 H, m, H-2b), 1.64 (1 H, m, H-15a), 1.63 (2 H, m, H-4b and H-7b), 1.61 (1 H, m, H-16a), 1.40 (1 H, dt, J ¼ 3.8 and 12.4 Hz, H-12a), 1.29 (2 H, m, H-6), 1.28 (1 H, m, H-11b), 1.27 (1 H, m, H-8), 1.17 (1 H, m, H-15b), 1.12 (1 H, m, H-14), 1.10 (1 H, m, H-1a), 1.07 (1 H, m, H-9), 1.06 (1 H, m, H-4ab), 0.88 (1H, m, H-7a), 0.79 (3 H, s, H-19), 0.73 (1 H, ddd, J ¼ 12.2, 10.5 and 3.8 Hz, H-5), 0.59 (3 H, s, H-18); 13C NMR dC: 209.5 (C-20), 71.5 (C-3), 63.9 (C-17), 57.0 (C-14), 53.5 (C-5), 48.4 (C-9), 44.1 (C-13), 39.3 (C-12), 38.7 (C-10), 36.9 (C-4), 36.3 (C-1), 35.3 (C-8), 32.3 (C-7), 31.53 (C-2), 31.47 (C-21), 31.3 (C-6), 26.6 (C-4a), 24.4 (C-15), 22.8 (C-16), 21.8 (C-11), 13.8 (C-19), 13.4 (C-18); MS (EI) m/z (%): 332 (Mþ, 9), 314 (13), 43 (100); HRMS (ESI) m/z: 333.2784 (Mþ þ H, C22H37O2 requires 333.2788), 315.2677 (Mþ þ H e H2O, C22H35O requires 315.2682).
4.3. Receptor binding assay An in vitro test, based on binding [35S]-tert-butyl-bicyclo[2.2.2] phosphorothionate to receptor of g-aminobutyric acid (GABAA), was used. Membranes were isolated from whole brains of adult male Wistar rats and re-suspended in a buffer (20 mM KH2PO4, 200 mM KCl, pH 7.4) [17]. Aliquots were incubated with 2 nM [35S]tert-butyl-bicyclo[2.2.2]phosphorothionate (TBPS, Perkin Elmer), 1 mM GABA and 1 nM e 10 mM steroids (added in DMSO solution) for 60 min at 37 C. The non-specific binding was estimated using
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200 mM picrotoxin. The results were related to the control samples containing DMSO (no steroid added) and expressed in percentage. Acknowledgements This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (PICT-00727), Universidad de Buenos Aires, CONICET (Argentina)-CSAV (Czech Republic) and Grant Agency of the Czech Republic (GACR203/08/1498 and Z40550506). References [1] D. Belelli, J. Lambert, Neurosteroids: endogenous regulators of the GABAA receptor. Nat. Rev. Neurosci. 6 (2005) 565e575. [2] B.O. Dubrosky, Steroids, neuroactive steroids and neurosteroids in psychopathology. Prog. Neuro-Psychoph. Biol. Psyc. 29 (2005) 169e192. [3] G. Akk, D.F. Covey, A.S. Evers, J.H. Steinbach, C.F. Zorumski, S. Mennerick, Mechanisms of neurosteroid interactions with GABAA receptors. Pharmacol. Therapeut. 116 (2007) 35e57. [4] A.S. Veleiro, G. Burton, Structureeactivity relationships of neuroactive steroids acting on the GABAA receptor. Curr. Med. Chem. 16 (2009) 455e472. [5] D.F. Covey, Y. Hu, M.G. Bouley, K.D. Holland, N.T. Rodgers-Neame, K. E. Isenberg, C.F. Zorumski, Modulation of GABAA receptor function by benz[e] indenes and phenanthrenes. J. Med. Chem. 36 (1993) 627e630. [6] M. Han, Y. Hu, Ch.F. Zorumski, D.F. Covey, Neurosteroid analogues. 3. The synthesis and electrophysiological evaluation of benz[e]indene congeners of neuroactive steroids having the 5b-configuration. J. Med. Chem. 38 (1995) 4548e4556. [7] P. Li, D.F. Covey, J.H. Steinbach, G. Akk, Dual potentiating and inhibitory actions of the benz[e]indene neurosteroid analog on recombinant a1b2g2 GABAA receptor. Mol. Pharmacol. 69 (2006) 2015e2016. [8] P.H. Di Chenna, M.V. Dansey, A.A. Ghini, G. Burton, Rearrangement of 4b,5bmethylenepregnanes: a simple approach to A-homopregnanes and 5bmethylpregnanes. Arkivoc xii (2005) 154e162. [9] Y. Dan, Ketone-catalyzed asymmetric epoxidation reactions. Acc. Chem. Res. 37 (2004) 497e505. [10] C. Annese, L. D’Accolti, A. Dinoi, C. Fusco, R. Gandolfi, R. Curci, Concerning the reactivity of dioxiranes. Observations from experiments and theory. J. Am. Chem. Soc. 130 (2008) 1197e1204. [11] A.K. Bose, W.A. Hoffman, M.S. Manhas, Steroids. IX. Facile inversion of unhindered sterol configuration. Tetrahedron Lett. (1973) 1619e1622. [12] J.R. Herr, A whirlwind tour of current Mitsunobu Chemistry, Albany Molecular Research, Inc. Tech. Rep. 3 (1999) 1e36. [13] J.W. Blunt, J.B. Stothers, 13C NMR Spectra of steroids-A survey and commentary. Org. Magn. Res. 9 (1977) 439e464. [14] W.B. Im, D.P. Blakeman, Correlation between gamma-aminobutyric acidA receptor ligand-induced changes in t-butylbicyclophosphoro [35S]thionate binding and 36Cl uptake in rat cerebrocortical membranes. Mol. Pharmacol. 39 (1991) 394e398. [15] Z.-X. Wang, Y. Tu, M. Frohn, J. Zhang, Y. Shi, An efficient catalytic asymmetric epoxidation method. J. Am. Chem. Soc. 119 (1997) 11224e11235. [16] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J. R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J. M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian, J.B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J. J. Dannenberg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M. W. Wong, C. Gonzalez, J.A. Pople, GAUSSIAN 03, Revision B.05. Gaussian, Inc., Pittsburgh, PA, 2003. [17] B. Slavíková, A. Kasal, H. Chodounská, Z. Kristofíková, 3a-Fluoro analogues of allopregnanolone and their binding to GABAA receptors. Coll. Czech. Chem. Commun. 67 (2002) 30e46.
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and GABAA receptor activity of A-homo analogues of neuroactive steroids María V. Dansey a, Pablo H. Di Chenna a, Adriana S. Veleiro a, Zdena Kristofíková b, Hana Chodounska c, Alexander Kasal c, Gerardo Burton a, * a
Departamento de Química Orgánica and UMYMFOR (CONICET-FCEN), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EGA Buenos Aires, Argentina Prague Psychiatric Centre, Ústavní 91, 181 03 Prague 8, Czech Republic c Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Fleming Square 2, CZ166 10 Prague 6, Czech Republic b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 November 2009 Received in revised form 17 February 2010 Accepted 26 March 2010 Available online 3 April 2010
A procedure is described for the preparation of A-homo-5-pregnenes via an acid catalyzed rearrangement of cyclopropylcarbinols assisted by microwave irradiation. 3a-Hydroxy and 4a-hydroxy-A-homo-5pregnen-20-one, analogues of the neuroactive steroid allopregnanolone, were obtained by means of a regioselective epoxidation of a double bond in the expanded A-ring, using a fructose-derived chiral ketone as catalyst and oxone as oxidant. Although both these compounds were marginally active in inhibiting TBPS binding to GABAA receptors, 3b-hydroxy-A-homo-5-pregnen-20-one was almost as active as allopregnanolone. Reduction of the double bond of the latter compound resulted in a ten fold loss of activity. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: A-homopregnane Neurosteroid g-Aminobutyric acid GABAA receptor
1. Introduction The term “neuroactive steroid” (NAS) refers to steroids which, independent of their origin, are capable of modifying neural activities. It is now demonstrated that these neuroactive steroids positively or negatively modulate the function of members of the ligand-gated ion channel receptor superfamily [1e4]. Most of these studies have focused on their positive allosteric actions on g-amino butyric acid type A receptor (GABAA receptor) as those elicited by the endogenous steroids allopregnanolone (1) and pregnanolone (2). The physiological and pharmacological actions of neuroactive steroids are topics of widespread interest, since they have shown to be potent anticonvulsants, anxiolytics, and antistress agents as well as to possess sedative, hypnotic, and anesthetic activities. Structure-activity relationship studies of neuroactive steroids at GABAA receptors [4] have established a pharmacophore for positive modulation of the receptor by steroids, consisting of a hydrogen bond accepting group (such as COCH3 or CN) in a pseudoequatorial configuration at the 17b position and a hydrogen bond donating hydroxyl group in the 3a configuration.
* Corresponding author. Fax: þ54 11 45763385. E-mail address: [email protected] (G. Burton). 0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2010.03.037
The importance of the steric constraint imposed on the 3ahydroxyl group by the steroid A ring for GABAergic activities was examined by Covey and coworkers, who prepared a series of nonsteroidal analogues of 1 and 2 that mimicked parts of the steroid nucleus [5,6]. Among these analogues, perhydro benz[e] indenes as 3 and 4 were potent modulators of GABAA receptor function with certain analogues of 4 (with a modified side chain) displaying both potentiating and inhibitory actions [7]. Perhydro benz[e]indenes are steroidelike molecules in which the A-ring has been replaced by an open chain of appropriate length, giving the molecule considerable flexibility at the position originally occupied by the critical 3a hydrogen bond donor. The greater flexibility of benz[e]indenes would allow the 3-hydroxy group to mimic steroids having either a 3a or a 3b hydroxyl, thus being able to bind to the potentiating and the inhibitory sites on the GABAA receptor [3]. Consequently, those studies demonstrated that GABAergic activity does not require the 3a-hydroxyl group to be kept in a fixed position by a rigid A ring. To further explore these effects, we envisaged that a more controlled conformational mobility of the allopregnanolone A ring could be obtained by its expansion to a seven membered ring. Also the additional carbon in the resulting A-homopregnanes would allow further variations in the position of the A ring hydroxyl (e.g. 5 and 6).
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M.V. Dansey et al. / European Journal of Medicinal Chemistry 45 (2010) 3063e3069 Table 1 Formation of the A-homo steroid 8 from the cyclopropyl alcohol 7 via cationic rearrangement. Entry 1 2 3 4 5
Catalyst BF3-éter/CH2Cl2 Br2Zn/THF AlCl3/THF Br2Zn/THF AlCl3/THF
Conditions
0 C, 10 min 25 C, 28 hs 25 C, 3.5 hs MW 120 C, 10 min MW 65 C, 10 min
Yield (%)a 70b NDc NDc 87 80
ND: not determined. MW: microwave irradiation. a After chromatographic purification. b Data taken from ref. [8]. c Complex mixture.
2. Results and discussion 2.1. Chemistry In a previous publication we showed that treatment of cyclopropyl alcohol 7 with BF3-Et2O in dichloromethane gave A-homopregnadiene 8 in moderate yield (Scheme 1; Table 1, entry 1) [8]. Further attempts to improve the yield of this rearrangement using either zinc bromide or aluminum chloride as catalysts at room temperature were unsuccesful as the reaction was slow and gave complex mixtures of byproducts (Table 1, entries 3 and 4). However, when these reactions were carried out under microwave irradiation (MW) very good to excellent yields of the homosteroid (8) were obtained (entries 5 and 6). Treatment of the latter compound with LiAlH4 in THF gave alcohol 9. With the A-homosteroid 9 in hand, we focused our attention on the regio and stereoselective functionalization of the D3double bond. Epoxidation with m-chloroperbenzoic acid (MCPBA) gave a mixture of the undesired 5,6-epoxide and the 3,4:5,6diepoxide. Since this reaction was electronically favored at the most substituted D5-olefin, we turned to dioxirane mediated epoxidation, a highly efficient and stereospecific method towards both electron-rich and electron-deficient olefins [9,10]. Taking into account that the D5-olefin was sterically hindered, we used the chiral and bulky fructose-derived ketone 10 as catalyst and potassium monopersulfate (Oxone) as oxidant. Under these conditions, the 3b,4b-epoxide 11 was obtained as the only product in 56% yield after recovery of unreacted material (Scheme 2). The stereospecific b-epoxidation of diene 9 may be explained by the bent conformation of the ring A towards the a face in this compound [8]. Confirmation of the stereochemistry of epoxide 11 came from NMR data and molecular modelling of the A ring alcohols obtained by reductive cleavage. Treatment of epoxide 11 with LiAlH4 gave a 9:2 mixture of 3b-hydroxy (12) and 4b-hydroxy (13) A-homopregnenes (77% yield) that could be separated by flash chromatography. The 1H NMR spectrum of diol 13 showed the resonance of H-4 at d 3.54 as a triplet of triplets (J ¼ 10.2 and 5.0 Hz) indicating an axial orientation of this
Scheme 1. Reagents and conditions: (a) see Table 1. (b) i. LiAlH4, THF ii. 1 N HCl.
hydrogen. A strong correlation between H-4 and H-6 was observed in the NOESY spectrum; molecular modelling of all conformers of the seven membered A-ring in 13 showed that this was only possible in the most stable conformer of the 4b-alcohol (Fig. 1a). The b orientation of the 4-hydroxyl confirmed the b stereochemistry of epoxide 11 and hence of the hydroxyl at C-3 in 12. The 1H NMR spectrum of alcohol 12 showed an unresolved multiplet at d 4.05 (W1/2 ¼ 10.8 Hz) for H-3, typical of an equatorial hydrogen. To convert the 3b-oriented axial alcohol in 12 into the neurosteroid analogue 6, we first attempted an oxidation reduction sequence (Scheme 2). Thus diol 12 was oxidized with pyridinium
Scheme 2. Reagents and conditions: (a) ketone 10 (30% mol), Oxone, tetrabutylammonium acetate, K2CO3(aq), CH3CN/DME (1:2); (b) i. LiAlH4, THF ii. 1 M HCl; (c) PCC, BaCO3, MS 4Å, CH2Cl2; (d) 1 M K-Selectride, THF, 50 C.
M.V. Dansey et al. / European Journal of Medicinal Chemistry 45 (2010) 3063e3069
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Fig. 2. Most stable conformer of compound 18 from HF/6-31G(d,p) calculations, showing NOEs and equatorial/axial orientation of relevant hydrogens. Other hydrogens are not shown for clarity.
Fig. 1. Most stable conformers from HF/6-31G(d,p) calculations, of compounds 13 (a), 15 (b), 5 (c) and 6 (d), showing NOEs and equatorial/axial orientation of relevant hydrogens. Other hydrogens are not shown for clarity. O(3)eC(20) distances for 3ahydroxy steroids are 5: 9.99 Å; 6: 10.72 Å.
chlorochromate to the diketone 14 followed by regioselective reduction of the ring A ketone with K-selectride (THF, 50 C). However, the resulting alcohol (15) had the 1H and 13C resonances of the A-ring atoms identical to those of 12, in particular it showed H-3 as an unresolved multiplet at d 4.05 (W1/2 ¼ 10.6 Hz)
Scheme 3. Reagents and conditions: (a) PPh3, HCOOH, DIAD, THF; (b) i. PCC, BaCO3, MS 4Å, CH2Cl2; ii. HCl, methanol, CH2Cl2.
indicating an axial orientation of the 3-hydroxyl. The NOESY spectrum of 15 had strong correlations among hydrogens at d 2.28 (4ab-H), 1.79 (1b-H), and 0.92 (19-H) and between the 4aa-H (d 1.90) and 6-H (d 5.41). Hydrogens at positions 4ab and 1b were assigned as axially oriented from their large vicinal coupling constants with 4a-H (J ¼ 13.7 Hz) and 2a-H (J ¼ 10.9 Hz) respectively, thus the b-orientation of the axial substituent at C-3 was confirmed. Molecular modelling of all conformers of the seven membered A-ring in 15 showed that, in the most stable conformer, the observed NOEs and couplings were consistent with the b orientation for the 3-hydroxyl (Fig. 1b) in agreement with the proposed configuration of diol 12. When the above sequence was applied to diol 13, the regioselective reduction of the intermediate diketone 16 occurred from the b face of ring A, giving the 4a-hydroxy analogue 5 as the only product in 70% yield (Scheme 2). The inversion of the stereochemistry at C-4 was evident from the change in the H-4 resonance (compared to 13), observed as an unresolved multiplet at d 3.94 (W1/2 ¼ 17.3 Hz) indicative of an equatorial hydrogen. The NOESY spectrum of 5 did not show a correlation between H-4 and H-6, but showed strong correlations among hydrogens at d 1.05 (1b), d 2.46 (4ab) and d 0.94 (H-19) which were indicative of the axial orientation of H-1b and H-4ab. The NOE correlation of both these hydrogens with H-3b (d 1.36) confirmed also its b-axial position, and the a orientation of the axial hydroxyl at C-4 (Fig. 1c). In view of the above results, the conversion of diol 12 into the 3a alcohol 6 was carried out by inversion of the alcohol at C-3 using the Mitsunobu reaction (Scheme 3) [11,12]. Thus, treatment of 12 with DIAD/Ph3P/HCO2H gave the 3a-formyloxy pregnane 17. Oxidation of 17 with pyridinium chlorochromate, followed by mild hydrolysis with potassium carbonate in methanol gave the target compound 6 (40% yield from 12). The inversion of stereochemistry at C-3 was evident from the change in the H-3 resonance (compared to 12 and 15) observed as a triple double doublet, with large axial-axial couplings with H-2a and H-4a (J ¼ 10.5 Hz), and smaller equatorial-axial couplings with H-2b and H-4b (J ¼ 3.7 and 5.5 Hz). The NOESY spectrum of 6 showed strong correlations of H-3 with H-1b (d 1.18), H-2b (1.64), H-4b (d 2.19) and H-4ab (1.99). The latter hydrogen and H-1b also had NOE correlations with H-19 (d 0.88) confirming the a orientation of the hydroxyl at C-3 (Fig. 1d). Hydrogenation of compound 15 gave the reduced 5a-pregnane 18 with good stereoselectivity (5a/5b 9:1 determined by 1H NMR of the crude reduction product). The stereochemistry at position 5 in compound 18 was inferred from the 13C NMR data. Thus, the C-19 resonance at d 13.8 for the 5a isomer was especially indicative of an A/B trans-fused steroid in comparison with the C-19 resonance at d 21.5 for the 5b isomer (observed as a minor component in the 13C NMR spectrum of the crude reduction product).[13] The NOESY spectrum of 18 showed correlations between H-5 (d 0.73) and hydrogens at positions 1a (d 1.10), 7a (d 0.88) and 9a (d 1.07). No
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correlation was observed between H-19 and H-5, confirming the a orientation of the hydrogen at C-5. The H-3 resonance in 18 appeared at d 3.94 as a double double triplet (J ¼ 8.8, 3.6 and 5.7 Hz) in contrast with the unresolved multiplet observed for compound 15, indicating a major change in the conformation of ring A. Fig. 2 shows the most stable conformer of 18 (from HF/6-31G(d,p) calculations), and the observed NOEs in good agreement with the proposed structure.
Fig. 3. Superposition of conformer 2 of A-homopregnane 15 (blue) and pregnanolone (2) (white) from HF/6-31G(d,p) calculations. Confomer 2 is 3.30 kcal/mol less stable than the most stable conformer of 15. Overlay corresponds to best fit for carbons 20, 16, 13, 11, 10 and 8 and O(3) (RMS error 0,22 Å); O(3)eC(20) distances are 2: 9.61 Å; 15: 9.93 Å.
3. Conclusion
2.2. Receptor binding studies GABAA receptor activity was evaluated by assaying the effect of the synthetic analogues 5, 6, 15 and 18 on the binding of [35S]-tertbutyl-bicyclo[2.2.2]phosphorothionate (TBPS). The binding of this convulsant in the presence of GABA closely reflects the functional state of GABAA receptors and may be useful for characterization of allosteric interactions between various sites on the receptor [4,14]. Allopregnanolone (1) was used as a positive control to check the viability of the methods. As shown in Table 2, compounds 5 and 6 with an a-oriented hydroxyl in ring A had a IC50 in the micromolar range; ab initio calculations show that both compounds adopt an overall bent conformation that resembles that of pregnanolone (2) (Fig. 1c and d), however attempts to overlay the structures of 5 and 6 with 2 resulted in a poor overall fit. Interestingly the 3b-alcohol 15 displayed an IC50 similar to that of allopregnanolone (1) but with a lower maximal inhibition. Although the most stable conformer of 15 has the 3-hydroxyl in an opposite orientation compared to allopregnanolone (1) and pregnanolone (2) (Fig. 1b), ab initio calculations showed two other conformers within 3.43 kcal/mol of the most stable one. One of these conformers, had an excellent fit with pregnanolone (Fig. 3), the 3-hydroxyl and the C-17 side chain occupying positions close in space for both compounds. Further support to this observation came from reduction of the 5,6 double bond in 15 to give 3b-alcohol 18. In this compound, the presence of the 5a-H and a sp3 carbon at position 5 reduces the flexibility of the A ring and precludes compound 18 from adopting a conformation smilar to that of pregnanolone. When compound 18 was assayed for its effect on TBPS binding, a ten fold loss of activity was observed.
Table 2 Inhibition of binding of [35S]-tert-butylbicyclo[2.2.2]phosporothionate ([35S]TBPS) to membranes from rat cerebellum by A-homo steroids 5, 6, 15 and 18. Compound
Imax (%)a
IC50 (nM)b
4a-hydroxy-A-homo-5-pregnen-20-one (5) 3a-hydroxy-A-homo-5-pregnen-20-one (6) 3b-hydroxy-A-homo-5-pregnen-20-one (15) 3b-hydroxy-A-homo-5aH-pregnan-20-one (18) Allopregnanolone (1)c
50.7 13.7 75.6 17.3 50.0 10.6 46.2 15.4 79.6 4.8
800 21.8 1200 553 100 55 1000 20 80 23
a b c
The maximal suppression of the binding. The steroid concentration producing a half-maximal inhibition. Data taken from ref. [17].
A series of four A-homo steroids related to the neurosteroids pregnanolone and allopreganolone have been synthesized and their GABAA receptor activity was evaluated in vitro. The conformational mobility of the seven-membered A-ring combined with the enhanced flexibility at the A/B ring junction introduced by the 5,6 double bond in ring B, would allow the 3b-hydroxy steroid 15 to mimic the pregnanolone molecule with a minor energy penalty, thus attaining an activity comparable to the natural neuroactive steroids.
4. Experimental 4.1. General Mps were taken on a Fisher-Johns apparatus and are uncorrected. IR spectra were recorded in thin films using KBr disks on a Nicolet Magna 550 FT-IR spectrophotometer, values are given in cm1. 1H and 13C NMR spectra were recorded on a Bruker Avance II 500 at 500.13 and 125.77 MHz respectively, in deuterochloroform. Chemical shifts are given in ppm downfield from TMS as internal standard, J values are given in Hz. Multiplicity determinations and 2D spectra (COSY, HSQC and HMBC) were obtained using standard Bruker software. The electron impact mass spectra (MS) were collected on a Shimadzu QP-5000 mass spectrometer at 70 eV by direct inlet. High resolution mass spectra (HRMS) were measured on an Agilent LCTOF, high resolution TOF analyzer or a Bruker micrOTOF-Q II spectrometer with ESI ionization. Elemental analysis was performed on an EAI Exeter Analytical, Inc. CE-440 apparatus. Microwave assisted reactions were carried out on a CEM Discover reactor, mode Discover (closed vessel, Power max: on). Vacuum liquid chromatography (VLC) and column flash chromatography were carried out on silica gel 60-G (Merck) and silica gel S 0.040e0.063 mm respectively. Thin layer chromatography (tlc) analysis was performed on silica gel 60 F254 (0.2 mm thick). The homogeneity of all compounds was confirmed by thin layer chromatography. Solvents were evaporated at reduced pressure and ca. 40e50 C. 3b-Hydroxy-20b-acetyloxy-4b,5b-methylenepregnane (7) was obtained from progesterone following the procedure previously described by us [8]. The ketone catalyst 10 was prepared from fructose, following the procedure described by Yian Shi et al. [15]. Geometry optimizations were carried out with the ab-initio quantum chemistry program GAUSSIAN 03 and the HF/6-31G(d,p) basis set [16].
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4.2. Chemical synthesis 4.2.1. A-homopregna-3,5-dien-20b-yl acetate (8) Method A: 3b-Hydroxy-4b,5b-methylenepregnan-20b-yl acetate (7) (0.030 g, 0.08 mmol) was dissolved in dry tetrahydrofuran (3 mL) under argon atmosphere. AlCl3 (0.063 g, 0.47 mmol) was added and the mixture was stirred in a microwave reactor at 65 C, 100 psi and 300 W for 10 min. The reaction mixture was diluted with aqueous NaHCO3 (5%) and extracted with dichloromethane. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification by flash chromatography (hexane-ethyl acetate, 97.5:2.5) gave diene 8 (0.023 g, 80%) as an amorphous solid identical (tlc, NMR) to that described previously [8]. Method B: 3b-Hydroxy-4b,5b-methylenepregnan-20b-yl acetate (7) (0.030 g, 0.08 mmol) was dissolved in dry tetrahydrofuran (3 mL) under argon atmosphere. ZnBr2 (0.120 g, 0.53 mmol) was added and the mixture was stirred in a microwave reactor at 120 C, 100 psi and 300 W for 10 minutes. Extractive work-up as above, gave diene 8 (0.025 g, 87%) as an amorphous solid identical (tlc, NMR) to that described previously [8]. 4.2.2. 20b-Hydroxy-A-homopregna-3,5-diene (9) To a solution of acetate 8 (0.120 g, 0.337 mmol) in diethyl ether (5 mL) under nitrogen atmosphere was added LiAlH4 (0.038 g, 0.99 mmol). After stirring for 1 h at room temperature HCl (1 N, 1 mL) was added, the mixture was poured onto an aqueous saturated solution of sodium and potassium tartrate and extracted with dichloromethane. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification by flash chromatography gave alcohol 9 (0.078 g, 74%) as a white crystalline solid identical (tlc, NMR) to that described previously [8]. 4.2.3. 3b,4b-Epoxy-20b-hydroxy-A-homo-5-pregnene (11) To a solution of compound 9 (0.250 g, 0.795 mmol) in acetonitrileedimethoxyethane (10.5 mL, 1:2, v/v) were succesively added ketone 10 (0.065 g, 0.252 mmol), tetrabutylammonium acetate (0.0046 mg, 0.028 mmol) and Na2(EDTA) (0.0013 g, 0.004 mmol). A solution of Oxone (1.312 g, 2.1 mmol) in aqueous Na2(EDTA) (4 104 M, 3.7 mL) and a solution of K2CO3 (0.987 g, 7.15 mmol) in water (3.7 mL), were then added simultaneously to the stirred mixture. Another 3.7 mL of each solution was added using a dual syringe pump (4 mL/h). Stirring was continued at room temperature for 30 min, the reaction was poured into HCl (1 N, 7.15 mL), and extracted with dichloromethane. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification by flash chromatography (hexane-ethyl acetate, 9:1) gave unreacted 9 (0.100 g, 40%) and epoxide 11 (0.088 g, 35%) as a white solid; mp 179e181 C (from hexaneeethyl acetate); Anal. calcd. for C22H34O2: C 79.95 H 10.4 found C 79.6 H 10.7; nmax (KBr, cm1) 3413, 2941, 2868, 1117, 972 and 881; 1H NMR dH: 5.38 (1 H, dd, J ¼ 4.2 and 1.2 Hz, H-6), 3.74 (1 H, m, H-20), 3.14 (1 H, m, H-4), 2.96 (1 H, m, H-3), 2.48 (1 H, dd, J ¼ 14.1 and 8.2 Hz, H-4aa), 2.36 (1 H, br d, J ¼ 14.1 Hz, H-4ab), 2.08 (1 H, dt, J ¼ 12.5 and 3.4 Hz, H-12b), 2.00 (1 H, m, H-7b), 1.95 (1 H, m, H-2a), 1.85 (1 H, m, H-2b), 1.67 (1 H, m, H-16b), 1.65 (1 H, m, H-15a), 1.57 (1 H, m, H-7a), 1.56 (1 H, m, H-8), 1.52 (1 H, m, H-11a), 1.41 (1 H, m, H-11b), 1.35 (3 H, m, H-1 and H-17), 1.27 (1 H, m, H-12a), 1.20 (1 H, m, H16a), 1.15 (3 H, d, J ¼ 6.0 Hz, H-21), 1.14 (1 H, m, H-15b), 1.14 (1 H, m, H-9), 1.08 (1 H, m, H-14), 0.87 (3 H, s, H-19), 0.76 (3 H, s, H-18); 13C NMR dC: 141.0 (C-5), 123.8 (C-6), 70.6 (C-20), 58.5 (C-17), 56.5 (C-14 and C-9), 55.6 (C-4), 55.3 (C-3), 42.2 (C-13), 40.3 (C-10), 40.0 (C12), 32.6 (C-4a), 31.6 (C-8 and C-7), 25.8 (C-1), 25.7 (C-16), 24.5 (C15), 23.7 (C-21), 23.2 (C-19), 23.0 (C-2), 21.3 (C-11), 12.5 (C-18); MS
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(EI) m/z (%): 330 (Mþ, 3), 315 (2), 207 (5), 189 (11), 163 (9), 91 (18), 55 (24), 45 (100). 4.2.4. 3b,20b-dihydroxy-A-homo-5-pregnene (12) and 4b,20bdihydroxy-A-homo-5-pregnene (13) To a stirred solution of epoxide 11 (0.330 g, 1 mmol) in dry tetrahydrofuran (33.5 mL) was added LiAlH4 (0.015 g, 1.6 mmol) under nitrogen atmosphere. After stirring for 20 min at room temperature, the reaction mixture was neutralized with 1 N HCl, poured onto an aqueous saturated solution of sodium and potassium tartrate and extracted with ethyl acetate. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification by flash chromatography (hexane-ethyl acetate) gave alcohols 12 (0.212 g, 63%) and 13 (0.047 g, 14%). Compound 12, amorphous solid; Anal. calcd. for C22H36O2: C 79.5 H 10.9 found C 79.7 H 10.8; nmax (KBr, cm1) 3388, 2954, 1486, 1052 and 749; 1H NMR dH: 5.41 (1 H, dd, J ¼ 5.8 and 2.2 Hz, H-6), 4.05 (1 H, bs, W1/2 ¼ 10.8 Hz, H-3), 3.75 (1 H, m, H-20), 2.30 (1 H, m, H-4ab), 2.09 (1 H, dt, J ¼ 12.6 and 3.4 Hz, H-12b), 2.02 (1H, dd, J ¼ 12.1 and 5.2 Hz, H-7b), 1.93 (1 H, m, H-4b), 1.92 (1 H, m, H-4aa), 1.76 (1 H, dd, J ¼ 15.0 and 11.2 Hz, H-1b), 1.69 (1 H, m, H-16b), 1.65 (1 H, m, H-15a), 1.63 (1 H, m, H-2b), 1.60 (1 H, m, H-11a), 1.59 (1 H, m, H-8), 1.58 (1 H, m, H-7a), 1.53 (1 H, m, H-1a), 1.50 (1 H, m, H-12a), 1.49 (1 H, m, H-4a), 1.46 (1 H, m, H-2a), 1.45 (1 H, m, H-11b), 1.35 (2 H, m, H-17), 1.22 (1 H, m, H-15b), 1.20 (1 H, m, H-16a), 1.16 (1 H, m, H-9), 1.15 (3 H, d, J ¼ 6.2 Hz, H-21), 1.08 (1 H, m, H-14), 0.93 (3 H, m, H-19), 0.79 (3 H, m, H-18); 13C NMR dC: 146.9 (C-5), 122.6 (C-6), 70.6 (C-20), 68.0 (C-3), 58.5 (C-17), 56.7 (C-14), 44.1 (C-9), 42.3 (C-13), 40.3 (C-4), 40.1 (C-12), 40.05 (C-10), 31.8 (C-8), 31.6 (C-7), 29.5 (C1), 26.9 (C-4a), 26.6 (C-2), 25.8 (C-16), 24.5 (C-15), 23.8 (C-19), 23.6 (C-21), 21.5 (C-11), 12.5 (C-18); MS (EI) m/z (%): 332 (Mþ, 19), 314 (10), 286 (23), 233 (46), 189 (100), 163 (45), 121 (48) 95 (48); HRMS (ESI) m/z: 333.2790 (Mþ þ H, C22H37O2 requires 333.2788). Compound 13 white solid; mp 179e180 C (from hexane-ethyl acetate); nmax (KBr, cm1) 3263, 2927, 1269, 1098, 1038, 1019 and 808; 1H NMR dH 5.52 (1 H, dd, J ¼ 5.5 and 1.8 Hz, H-6), 3.74 (1 H, dq, J ¼ 9.8 and 6.1 Hz, H-20), 3.54 (1 H, tt, J ¼ 10.2 and 5.0 Hz, H-4), 2.24 (1 H, dd, J ¼ 13.0 and 5.0 Hz, H4aa), 2.20 (1H, br t, J ¼ 12.4 Hz, H4ab), 2.07 (2 H, m, H-3a), 2.06 (1H, dt, J ¼ 12.5 and 3.5 Hz, H-12b), 2.03 (1H, m, H-7b), 1.94 (1 H, dd, J ¼ 14.5 and 8.8 Hz, H-1a), 1.67 (1 H, m, H16b),1.63 (1 H, m, H-15a),1.55 (2 H, m, H-8 and H-7a),1.54 (1 H, m, H11a), 1.43 (1 H, m, H-2a), 1.41 (1 H, m, H-11b), 1.33 (1 H, br q, J ¼ 9.8 Hz, H-17),1.25 (1 H, m, H-12a),1.21 (1 H, m, H-3b),1.17 (1 H, m, H-16a),1.16 (1 H, m, H-1b),1.14 (1 H, m, H-15b),1.14 (3 H, d, J ¼ 6.1 Hz, H-21), 1.08 (2 H, m, H-9 and H-2b), 1.06 (1 H, m, H-14), 0.91 (3 H, s, H19), 0.78 (3 H, s, H-18); 13C NMR dC: 140.7 (C-5), 125.4 (C-6), 76.1 (C4), 70.6 (C-20), 58.5 (C-17), 56.6 (C-14), 44.0 (C-9), 42.3 (C-13), 42.0 (C-4a), 40.4 (C-3), 40.1 (C-12), 40.0 (C-10), 35.9 (C-1), 31.6 (C-8), 31.5 (C-7), 25.7 (C-16), 24.4 (C-15), 23.7 (C-19), 23.6 (C-21), 21.4 (C-11), 18.8 (C-2), 12.5 (C-18); MS (EI) m/z (%): 332 (Mþ, 29), 314 (24), 233 (65), 189 (100), 163 (51), 119 (69), 105 (74), 55(83), 45 (91); HRMS (ESI) m/z: 333.2784 (Mþ þ H, C22H37O2 requires 333.2788). 4.2.5. A-homo-5-pregnene-3,20-dione (14) A solution of alcohol 12 (0.059 g, 0.18 mmol) in dichloromethane (19 mL) was added to a stirred suspension of PCC (0.364 g, 1.65 mmol), BaCO3 (0.101 g, 0.52 mmol) and activated 4Å molecular sieves (0.144 g) in dry dichloromethane (10 mL). After stirring at room temperature for 4 h, celite (1 g) was added and the solvent was removed under reduced pressure. The mixture was percholated through a short silica gel column (hexaneeethyl acetate) and the residue obtained after evaporation of the solvent was purified by flash chromatography (hexaneeethyl acetate, 95:5) to give diketone 14 (0.039 g, 66%) as a white solid; mp 166-167 C (from hexane-ethyl acetate); nmax (KBr, cm1) 2934, 1703, 1354 and 1236;
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1
H NMR dH 5.57 (1 H, dd, J ¼ 5.2 and 2.0 Hz, H-6), 2.63 (1 H, dt, J ¼ 14.7 and 4.7 Hz, H-4b), 2.56 (1 H, t, J ¼ 8.7 Hz, C-17), 2.45 (1 H, m, H-4ab), 2.36 (1 H, m, H-4a), 2.32 (2 H, m, H-2), 2.19 (1 H, m, H-16b), 2.16 (1 H, m, H-4aa), 2.13 (3 H, s, H-21), 2.08 (1 H, m, H-12b), 2.06 (1 H, m, H-7b), 1.76 (2 H, m, H-1), 1.70 (1 H, m, H-15a), 1.69 (1 H, m, H16a), 1.63 (1 H, m, H-11a), 1.61 (1 H, m, H-7a), 1.58 (1 H, m, H-8), 1.48 (1 H, m, H-12a), 1.47 (1 H, m, H-11b), 1.23 (1 H, m, H-14), 1.22 (1 H, m, H-9), 1.21 (1 H, m, H-15b), 1.00 (3 H, s, H-19), 0.65 (3 H, s, H-18); 13 C NMR dC: 213.1 (C-3), 209.4 (C-20), 142.7 (C-5), 125.2 (C-6), 63.6 (C-17), 57.1 (C-14), 44.6 (C-9), 44.1 (C-4), 43.9 (C-13), 38.9 (C-2 and C-10), 38.8 (C-12), 31.8 (C-8), 31.5 (C-21), 31.3 (C-7), 31.1 (C-1), 27.9 (C-4a), 24.4 (C-15), 22.9 (C-16), 22.0 (C-19), 21.5 (C-11), 13.3 (C-18); MS (EI) m/z (%): 328 (Mþ, 55), 310 (23), 300 (30), 205 (35), 119 (32), 105 (35), 55 (39), 43 (100); HRMS (ESI) m/z: 329.2476 (Mþ þ H, C22H33O2 requires 329.2475). 4.2.6. A-homo-5-pregnene-4,20-dione (16) Alcohol 13 (0.0086 g, 0.029 mmol) was oxidized with PCC (0.054 g, 0.245 mmol) following the procedure described for compound 12, to give diketone 16 (0.0064 g, 75%) as a white solid; mp 141-144 C (from hexane-ethyl acetate); nmax (KBr, cm1) 2918, 1701, 1541, 1508.1, 1456 and 1356; 1H NMR dH: 5.57 (1 H, dd, J ¼ 5.5 and 2.1 Hz, H-6), 3.26 (1 H, d, J ¼ 14.4 Hz, H-4aa), 2.84 (1 H, d, J ¼ 14.4 Hz, H-4ab), 2.62 (1 H, m, H-3a), 2.55 (1 H, t, J ¼ 9.1 Hz, H17), 2.22 (1 H, m, H-3b), 2.19 (1 H, m, H-16b), 2.13 (3 H, s, H-21), 2.09 (1 H, m, H-7b), 2.08 (1 H, m, H-12b), 1.87 (1 H, dd, J ¼ 13.0 and 7.1 Hz, H-1a), 1.68 (1 H, m, H-16a), 1.67 (1 H, m, H-15a), 1.66 (1 H, m, H-11a), 1.65 (1 H, m, H-2a), 1.63 (1 H, m, H-7a), 1.60 (1 H, m, H-8), 1.57 (2 H, m, H-2b and H-11b), 1.55 (1 H, m, H-1b), 1.48 (1 H, m, H12a), 1.30 (1 H, m, H-9), 1.25 (1 H, m, H-15b), 1.23 (1 H, m, H-14), 0.99 (3 H, s, H-19), 0.65 (3 H, s, H-18); 13C NMR dC: 210.7 (C-4), 209.5 (C-20), 137.2 (C-5), 127.1 (C-6), 63.6 (C-17), 57.1 (C-14), 48.6 (C-4a), 44.0 (C-9), 43.9 (C-13), 42.5 (C-3), 39.8 (C-10), 38.8 (C-12), 34.9 (C1), 31.7 (C-8), 31.6 (C-7), 31.5 (C-21), 24.4 (C-15), 22.9 (C-16), 22.7 (C-19), 21.4 (C-11), 18.6 (C-2), 13.3 (C-18); MS (EI) m/z (%): 328 (Mþ, 70), 205 (24), 187 (28), 105 (46), 85 (54), 55 (56), 43 (100); HRMS (ESI) m/z: 329.2475 (Mþ þ H, C22H33O2 requires 329.2475). 4.2.7. 3b-Hydroxy-A-homo-5-pregnen-20-one (15) A solution of K-Selectride in tetrahydrofuran (1 M, 0.22 mL, 0.22 mmol) was added to a stirred solution of diketone 14 (0.060 g, 0.183 mmol) in dry tetrahydrofuran (5 mL) at -50 C, under nitrogen atmosphere. After 30 min the reaction mixture was poured onto an aqueous ammonium chloride solution (5%) and extracted with dichloromethane. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification by flash chromatography (hexane-ethyl acetate, 85:15) gave the 3b-alcohol 15 (0.038 g, 63%) as a white solid; mp 151 153 C (from hexaneethyl acetate); Anal. calcd. for C22H34O2: C 79.95 H 10.4 found: C 79.9 H 10.4; nmax (KBr, cm1) 3329, 2934, 1710 and 1038; 1H NMR dH: 5.41 (1 H, dd, J ¼ 5.1 and 1.6 Hz, H-6), 4.05 (1 H, br s, W1/2 ¼ 10.6 Hz, H-3), 2.53 (1 H, t, J ¼ 9.0 Hz, H-17), 2.28 (1 H, dt, J ¼ 3.1 and 13.7 Hz, H-4ab), 2.19 (1 H, m, H-16b), 2.13 (3 H, s, H-21), 2.06 (1 H, m, H-12b), 2.05 (1 H, m, H-7b), 1.92 (1 H, m, H-4b), 1.90 (1 H, m, H-4aa), 1.79 (1 H, dd, J ¼ 15.2 and 10.9 Hz, H-1b), 1.68 (2 H, m, H-15a and H-11a), 1.67 (1 H, m, H-16a), 1.63 (1 H, m, H-2b), 1.57 (1 H, m, H-7a), 1.56 (1 H, m, H-8), 1.51 (1 H, dd, J ¼ 15.2 and 8.3 Hz, H-1a), 1.48 (1 H, m, H-4a), 1.46 (1 H, m, H-12a), 1.44 (1 H, m, H-11b), 1.43 (1 H, m, H-2a), 1.23 (1 H, m, H15b), 1.20 (1 H, m, H-14), 1.16 (1 H, m, H-9), 0.92 (3 H, s, H-19), 0.64 (3 H, s, H-18); 13C NMR dC: 209.6 (C-20), 146.8 (C-5), 122.4 (C-6), 67.8 (C3), 63.7 (C-17), 57.4 (C-14), 44.02 (C-13), 44.00 (C-9), 40.3 (C-4), 40.0 (C-10), 39.0 (C-12), 31.9 (C-8), 31.5 (C-21), 31.3 (C-7), 29.4 (C-1), 26.9 (C-4a), 26.6 (C-2), 24.3 (C-15), 23.7 (C-19), 22.8 (C-16), 21.6 (C-11), 13.8 (C-18); MS (EI) m/z (%): 330 (Mþ, 23), 312 (15), 302 (14), 231 (46), 205 (22), 187 (25), 121 (36), 43 (100).
4.2.8. 4a-Hydroxy-A-homo-5-pregnen-20-one (5) Diketone 16 (0.007 g, 0.021 mmol) was reduced with 1 M Kselectride in dry tetrahydrofuran (0.025 mL, 0.025 mmol) following the procedure described for compound 14, to give 5 (0.005 g, 70%) as an amorphous solid; Anal. calcd. for C22H34O2: C 79.95 H 10.4 found C 79.9 H 10.4; nmax (KBr, cm1) 2928, 1705, 1541 and 1458; 1H NMR dH: 5.52 (1 H, dd, J ¼ 4.9 and 1.9 Hz, H-6), 3.94 (1 H, br s, W1/2 ¼ 15.0 Hz, H-4), 2.54 (1 H, t, J ¼ 9.0 Hz, H-17), 2.46 (1 H, br d, J ¼ 13.6 Hz, H-4ab), 2.22 (1 H, dd, J ¼ 13.6 and 1.0 Hz, H-4aa), 2.20 (1 H, m, H-16b), 2.13 (3 H, s, H-21), 2.11 (1 H, dt, J ¼ 16.8 and 4.7 Hz, H-7b), 2.06 (1 H, dt, J ¼ 12.0 and 3.1 Hz, H-12b), 1.98 (1 H, dd, J ¼ 14.4 and 8.9 Hz, H-1a), 1.87 (1 H, br d, J ¼ 14.3 Hz, H-3a), 1.70 (2 H, m, H-15a and H-16a), 1.69 (2 H, m, H-11a and H-7a), 1.64 (1 H, m, H-8), 1.48 (1 H, dt, J ¼ 2.9 and 12.0 Hz, H-12a), 1.41 (1 H, m, H-11b), 1.36 (1 H, m, H-3b), 1.33 (1 H, m, H-2b), 1.25 (1 H, m, H-15b), 1.23 (1 H, m, H-14),1.20 (1 H, m, H-9), 1.12 (1 H, m, H-2a),1.05 (1 H, dd, J ¼ 14.4 and 10.0 Hz, H-1b), 0.94 (3 H, s, H19), 0.64 (3 H, s, H-18); 13C NMR dH: 209.6 (C-20),140.1 (C-5),126.7 (C6), 66.0 (C-4), 63.6 (C-17), 57.3 (C-14), 44.5 (C-10), 44.02 (C-13), 44.00 (C-9), 39.3 (C-3), 39.0 (C-12), 38.3 (C-4a), 36.7 (C-1), 31.8 (C-8), 31.5 (C-21), 30.9 (C-7), 24.3 (C-15), 24.0 (C-19), 22.9 (C-16), 21.6 (C-11),17.1 (C-2), 13.4 (C-18); MS (EI) m/z (%): 330 (Mþ, 1), 312 (5), 149 (23), 105 (21), 85 (27), 55 (19), 43 (100). 4.2.9. 3a-Hydroxy-A-homo-5-pregnen-20-one (6) A solution of diisopropyl azodicarboxylate (0.027 mL, 0.12 mmol) in dry tetrahydrofuran (0.116 mL) was added to a solution of diol 12 (0.020 g, 0.060 mmol), triphenylphosphine (0.032 g, 0.12 mmol) and formic acid (0.0048 mL, 0.12 mmol), in dry tetrahydrofuran (0.75 mL). After stirring for 20 h at room temperature, the reaction mixture was diluted with CH2Cl2 and silica gel (0.100 g) was added. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (hexane to hexaneeethyl acetate, 95:5), to give formate 17 (0.016 g, 71%) as an amorphous solid; 1H NMR dH: 5.49 (1 H, dd, J ¼ 4.8 and 1.6 Hz, H-6), 4.80 (1 H, m, H-3), 3.72 (1 H, m, H-20), 1.15 (3 H, d, J ¼ 6.2 Hz, H-21), 0.90 (3 H, s, H-19), 0.80 (3 H, s, H-18); 13C NMR dC: 70.6 (C-20), 145.4 (C-5), 123.9 (C-6), 37.3 (C-4), 58.5 (C-17), 56.6 (C-14), 44.2 (C-9), 42.2 (C-13), 27.7 (C-4a), 76.5 (C-3), 40.26 (C-10), 40.0 (C-12), 31.6 (C8), 31.5 (C-7), 30.8 (C-1), 28.7 (C-2), 25.7 (C-16), 24.4 (C-15), 23.6 (C21), 23.3 (C-19), 21.4 (C-11), 12.5 (C-18). A solution of formate 17 (0.015 g, 0.04 mmol) in dichloromethane (1.5 mL) was added to a stirred suspension of PCC 0.036 g, 0.165 mmol), BaCO3 (0.015 g, 0.080 mmol) and 4Å molecular sieves (0.030 g) in dry dichloromethane (1 mL). After stirring for 1 h at room temperature, celite (0.100 g) was added and the mixture was percholated through a silica gel column (hexaneeethyl acetate, 95:5), to give the 20ketone (0.009 g, 60%) as an amorphous solid; 1H NMR dH: 5.49 (1 H, dd, J ¼ 5.5 and 2.0 Hz, H-6), 4.84 (1 H, m, H-3), 2.12 (3 H, s, H-21), 0.89 (3 H, s, H-19), 0.63 (3 H, s, H-18); 13C NMR dC: 209.6 (C-20), 145.3 (C-5), 123.8 (C-6), 37.4 (C-4), 63.7 (C-17), 57.3 (C-14), 44.2 (C9), 44.0 (C-13), 27.8 (C-4a), 76.6 (C-3), 39.0 (C-12), 40.3 (C-10), 31.7 (C-8), 31.3 (C-7), 30.9 (C-1), 28.7 (C-2), 22.9 (C-16), 24.4 (C-15), 31.6 (C-21), 23.3 (C-19), 21.6 (C-11), 13.4 (C-18). To the 20-ketone obtained above (0.009 g, 0.02 mmol) in a mixture of dichloromethane (0.16 mL), methanol (0.53 mL) and water (0.04 mL), was added conc. HCl (0.078 mL, 0.94 mmol). After stirring for 1 h at room temperature, the reaction mixture was neutralized with aqueous NaHCO3 and extracted with dichloromethane. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. Purification by preparative tlc (hexane-ethyl acetate, 7:3) gave compound 6 (0.008 g, 100%) as a white solid; mp 165e170 C (from hexane-ethyl acetate); Anal. calcd. for C22H34O2: C 79.95 H 10.4 found C 79.8 H 10.4; nmax (KBr, cm1) 3473, 2928, 1697, 14589, 1043; 1H NMR dH: 5.47 (1 H, dd, J ¼ 5.0 and 2.0 Hz, H-6), 3.60 (1 H, tdd, J ¼ 10.5, 5.5 and 3.7 Hz, H-3),
M.V. Dansey et al. / European Journal of Medicinal Chemistry 45 (2010) 3063e3069
2.54 (1 H, t, J ¼ 8.4 Hz, H-17), 2.19 (2 H, m, H-16b and H-4b), 2.12 (3 H, s, H-21), 2.05 (2 H, m, H-12b and H-7b), 1.99 (2 H, m, H-4aa and H-4ab), 1.82 (1 H, dd, J ¼ 15.1 and 9.5 Hz, H-1a), 1.69 (1 H, m, H-15a), 1.66 (2 H, m, H-11a and H-16a), 1.64 (1 H, m, H-2b), 1.59 (1 H, m, H7a), 1.55 (1 H, m, H-8), 1.47 (1 H, dt, J ¼ 2.8 and 12.8 Hz, H-12a), 1.41 (1 H, dq, J ¼ 3.2 and 12.8 Hz, H-11b), 1.31 (1 H, dt, J ¼ 13.3 and 10.5 Hz, H-2a), 1.23 (1 H, m, H-15b), 1.22 (1 H, m, H-4a), 1.21 (1 H, m, H-14), 1.18 (1 H, dd, J ¼ 15.1 and 10.5 Hz, H-1b), 1.17 (1 H, m, H-9), 0.88 (3 H, s, H-19), 0.63 (3 H, s, H-18); 13C NMR dC: 209.6 (C-20), 145.9 (C-5), 123.3 (C-6), 74.9 (C-3), 63.7 (C-17), 57.3 (C-14), 44.2 (C9), 44.0 (C-13), 41.4 (C-4), 40.4 (C-10), 39.0 (C-12), 32.5 (C-2), 31.7 (C-8), 31.5 (C-21), 31.3 (C-7), 31.1 (C-1), 28.0 (C-4a), 22.9 (C-16), 24.3 (C-15), 23.3 (C-19), 21.6 (C-11), 13.3 (C-18); MS (EI) m/z (%): 330 (Mþ, 16), 312 (34), 284 (41), 231 (20), 187 (21), 91 (45), 79 (45), 43 (100); HRMS (ESI) m/z: 331.2633 (Mþ þ H, C22H35O2 requires 331.2632), 313.2528 (Mþ þ HeH2O, C22H33O requires 313.2526). 4.2.10. 3b-Hydroxy-A-homo-5aH-pregnan-20-one (18) 10% Palladium on carbon (0.060 g) was added to a solution of steroid 15 (0.020 g, 0.061 mmol) in ethyl acetate (5 mL). The suspension was hydrogenated for 22 h at 60 psi and 25 C, the reaction mixture was filtered through a silica gel column and the solvent was evaporated under reduced pressure. The residue (a 9:1 mixture of 5aH and 5bH steroids as determined by 1H NMR) was purified by preparative tlc (hexane-ethyl acetate, 8:2) to give homopregnane 18 (0.016 g, 80%) as a white solid; mp 148-150 C (from hexane-ethyl acetate); Anal. calcd. for C22H36O2: C 79.5 H 10.9 found C 79.4 H,10.7; nmax (KBr, cm1) 3421, 2926, 1695, 1541, 1458 and 1038; 1H NMR dH: 3.94 (1 H, ddt, J ¼ 8.8, 3.6 and 5.7 Hz, H-3), 2.51 (1 H, t, J ¼ 9.0 Hz, H-17), 2.14 (1 H, m, H-16b), 2.11 (3 H, s, H-21), 2.00 (1 H, dt, J ¼ 12.4 and 3.3 Hz, H-12b), 1.91 (1 H, m, H-4a), 1.75 (1 H, m, H-1b), 1.73 (1 H, m, H-2a), 1.69 (2 H, m, H-4aa and H-11a), 1.65 (1 H, m, H-2b), 1.64 (1 H, m, H-15a), 1.63 (2 H, m, H-4b and H-7b), 1.61 (1 H, m, H-16a), 1.40 (1 H, dt, J ¼ 3.8 and 12.4 Hz, H-12a), 1.29 (2 H, m, H-6), 1.28 (1 H, m, H-11b), 1.27 (1 H, m, H-8), 1.17 (1 H, m, H-15b), 1.12 (1 H, m, H-14), 1.10 (1 H, m, H-1a), 1.07 (1 H, m, H-9), 1.06 (1 H, m, H-4ab), 0.88 (1H, m, H-7a), 0.79 (3 H, s, H-19), 0.73 (1 H, ddd, J ¼ 12.2, 10.5 and 3.8 Hz, H-5), 0.59 (3 H, s, H-18); 13C NMR dC: 209.5 (C-20), 71.5 (C-3), 63.9 (C-17), 57.0 (C-14), 53.5 (C-5), 48.4 (C-9), 44.1 (C-13), 39.3 (C-12), 38.7 (C-10), 36.9 (C-4), 36.3 (C-1), 35.3 (C-8), 32.3 (C-7), 31.53 (C-2), 31.47 (C-21), 31.3 (C-6), 26.6 (C-4a), 24.4 (C-15), 22.8 (C-16), 21.8 (C-11), 13.8 (C-19), 13.4 (C-18); MS (EI) m/z (%): 332 (Mþ, 9), 314 (13), 43 (100); HRMS (ESI) m/z: 333.2784 (Mþ þ H, C22H37O2 requires 333.2788), 315.2677 (Mþ þ H e H2O, C22H35O requires 315.2682).
4.3. Receptor binding assay An in vitro test, based on binding [35S]-tert-butyl-bicyclo[2.2.2] phosphorothionate to receptor of g-aminobutyric acid (GABAA), was used. Membranes were isolated from whole brains of adult male Wistar rats and re-suspended in a buffer (20 mM KH2PO4, 200 mM KCl, pH 7.4) [17]. Aliquots were incubated with 2 nM [35S]tert-butyl-bicyclo[2.2.2]phosphorothionate (TBPS, Perkin Elmer), 1 mM GABA and 1 nM e 10 mM steroids (added in DMSO solution) for 60 min at 37 C. The non-specific binding was estimated using
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200 mM picrotoxin. The results were related to the control samples containing DMSO (no steroid added) and expressed in percentage. Acknowledgements This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (PICT-00727), Universidad de Buenos Aires, CONICET (Argentina)-CSAV (Czech Republic) and Grant Agency of the Czech Republic (GACR203/08/1498 and Z40550506). References [1] D. Belelli, J. Lambert, Neurosteroids: endogenous regulators of the GABAA receptor. Nat. Rev. Neurosci. 6 (2005) 565e575. [2] B.O. Dubrosky, Steroids, neuroactive steroids and neurosteroids in psychopathology. Prog. Neuro-Psychoph. Biol. Psyc. 29 (2005) 169e192. [3] G. Akk, D.F. Covey, A.S. Evers, J.H. Steinbach, C.F. Zorumski, S. Mennerick, Mechanisms of neurosteroid interactions with GABAA receptors. Pharmacol. Therapeut. 116 (2007) 35e57. [4] A.S. Veleiro, G. Burton, Structureeactivity relationships of neuroactive steroids acting on the GABAA receptor. Curr. Med. Chem. 16 (2009) 455e472. [5] D.F. Covey, Y. Hu, M.G. Bouley, K.D. Holland, N.T. Rodgers-Neame, K. E. Isenberg, C.F. Zorumski, Modulation of GABAA receptor function by benz[e] indenes and phenanthrenes. J. Med. Chem. 36 (1993) 627e630. [6] M. Han, Y. Hu, Ch.F. Zorumski, D.F. Covey, Neurosteroid analogues. 3. The synthesis and electrophysiological evaluation of benz[e]indene congeners of neuroactive steroids having the 5b-configuration. J. Med. Chem. 38 (1995) 4548e4556. [7] P. Li, D.F. Covey, J.H. Steinbach, G. Akk, Dual potentiating and inhibitory actions of the benz[e]indene neurosteroid analog on recombinant a1b2g2 GABAA receptor. Mol. Pharmacol. 69 (2006) 2015e2016. [8] P.H. Di Chenna, M.V. Dansey, A.A. Ghini, G. Burton, Rearrangement of 4b,5bmethylenepregnanes: a simple approach to A-homopregnanes and 5bmethylpregnanes. Arkivoc xii (2005) 154e162. [9] Y. Dan, Ketone-catalyzed asymmetric epoxidation reactions. Acc. Chem. Res. 37 (2004) 497e505. [10] C. Annese, L. D’Accolti, A. Dinoi, C. Fusco, R. Gandolfi, R. Curci, Concerning the reactivity of dioxiranes. Observations from experiments and theory. J. Am. Chem. Soc. 130 (2008) 1197e1204. [11] A.K. Bose, W.A. Hoffman, M.S. Manhas, Steroids. IX. Facile inversion of unhindered sterol configuration. Tetrahedron Lett. (1973) 1619e1622. [12] J.R. Herr, A whirlwind tour of current Mitsunobu Chemistry, Albany Molecular Research, Inc. Tech. Rep. 3 (1999) 1e36. [13] J.W. Blunt, J.B. Stothers, 13C NMR Spectra of steroids-A survey and commentary. Org. Magn. Res. 9 (1977) 439e464. [14] W.B. Im, D.P. Blakeman, Correlation between gamma-aminobutyric acidA receptor ligand-induced changes in t-butylbicyclophosphoro [35S]thionate binding and 36Cl uptake in rat cerebrocortical membranes. Mol. Pharmacol. 39 (1991) 394e398. [15] Z.-X. Wang, Y. Tu, M. Frohn, J. Zhang, Y. Shi, An efficient catalytic asymmetric epoxidation method. J. Am. Chem. Soc. 119 (1997) 11224e11235. [16] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J. R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J. M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian, J.B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J. J. Dannenberg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M. W. Wong, C. Gonzalez, J.A. Pople, GAUSSIAN 03, Revision B.05. Gaussian, Inc., Pittsburgh, PA, 2003. [17] B. Slavíková, A. Kasal, H. Chodounská, Z. Kristofíková, 3a-Fluoro analogues of allopregnanolone and their binding to GABAA receptors. Coll. Czech. Chem. Commun. 67 (2002) 30e46.