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New 5α-reductase inhibitors: In vitro and in vivo effects
Steroids 70 (2005) 217–224
New 5␣-reductase inhibitors: In vitro and in vivo effects V´ıctor P´erez-Ornelas a , Marisa Cabeza b, ∗ , Eugene Bratoeff a , Ivonne Heuze b , Mauricio S´anchez b , Elena Ram´ırez a , Elia Naranjo-Rodr´ıguez a b
a Department of Pharmacy, Faculty of Chemistry, National University of Mexico City, Mexico D.F., Mexico Departments of Biological Systems and Animal Production of Metropolitan University of M´exico Center Xochimilco, Calzada Del Hueso 1100, 04960 Mexico D.F., Mexico
Received 19 May 2004; received in revised form 13 October 2004; accepted 5 November 2004
Abstract The enzyme 5␣-reductase is responsible for the conversion of testosterone (T) to its more potent androgen dihydrotestosterone (DHT). This steroid had been implicated in androgen-dependent diseases such as: benign prostatic hyperplasia, prostate cancer, acne and androgenic alopecia. The inhibition of 5␣-reductase enzyme offers a potentially useful treatment for these diseases. In this study, we report the synthesis and pharmacological evaluation of several new 3-substituted pregna-4, 16-diene-6, 20-dione derivatives. These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological activity of the new steroidal derivatives was determined in vivo as well as in vitro experiments. In vivo experiments, the anti-androgenic effect of the steroids was demonstrated by the decrease of the weight of the prostate gland of gonadectomized hamster treated with T plus finasteride or the new steroids. The IC50 value of these steroids was determined by measuring the conversion of radio labeled T to DHT. The results of this study carried out with 5␣-reductase enzyme from hamster and human prostate showed that four of the six steroidal derivatives (5, 7, 9, 10) exhibited much higher 5␣-reductase inhibitory activity, as indicated by the IC50 values than the presently used Proscar 3 (finasteride). The comparison of the weight of the hamster’s prostate gland indicated that compound 5 had a comparable weight decrease as finasteride. The overall data of this study showed very clearly those compounds 5, 7, 9, 10 are good inhibitors for the 5␣-reductase enzyme. © 2005 Elsevier Inc. All rights reserved. Keywords: 5␣-Reductase; 5␣-Reductase inhibitors; Prostate; Pregnadiene derivatives; In vivo effect of 5␣-reductase inhibitors; Epoxide toxicity
1. Introduction The normal activity of the NADPH-dependent 5␣reductase enzyme (EC 1.3.99.5) maintains testosterone’s 1 (T) biological functions: anabolic actions and spermatogenesis of humans as well as the dihydrotestosterone 2 mediated effects such as, increased facial and body hair, acne, scalp hair recession, and prostate enlargement [1]. Abnormally high 5␣-reductase activity in humans results in excessively high DHT levels in peripheral tissues, which is implicated in the pathogenesis of prostate cancer, benign prostatic hyperplasia ∗
Corresponding author. Tel.: +52 55 5483 72 60; fax: +52 55 5483 72 60. E-mail address: [email protected] (M. Cabeza).
0039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2004.11.008
(BPH), acne, and male patter baldness [2,3], thus suggesting that both the enzyme 5␣-reductase and DHT play important physiological and pathological roles in humans. Therefore, the suppression of androgen action by 5␣-reductase inhibitors is a logical treatment for 5␣-reductase activity disorders. Furthermore, since the beginning of the last decade, two types of 5␣-reductase enzyme had been identified 1 and 2 [4,5]; the identification of these two isozymes opened the door for specific and selective inhibition. The most extensively studied class of 5␣-reductase inhibitors are the 4-azasteroids [6,7], which include the drug finasteride 3. This compound is the first 5␣-reductase inhibitor approved in the USA for the treatment of BPH. This drug has approximately a 100-fold greater affinity for type
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2–5␣-reductase, than for the type 1 enzyme. In humans, finasteride decreases prostatic DHT levels by 70–90% and reduces prostate size, while T tissue levels remain constant [8]. The use of finasteride demonstrated a sustained improvement in the treatment of androgen-dependent diseases and it also reduces the prostate specific antigen (PSA) levels [8]. Recently, our group synthesized several new progesterone derivatives that considerably decreased the prostate growth produced by T [9–12]. Since these compounds showed a high biological activity [9–12], in this paper, we describe the synthesis and pharmacological evaluation of six similar compounds (5–11) based on the progesterone skeleton as inhibitors of hamster and human 5␣-reductase enzyme.
2. Experimental 2.1. Chemical and radioactive material Solvents were laboratory grade or better. (1, 2, 6, 7–3 H) Testosterone [3 H] T specific activity: 95 Ci/mmol was provided by New England Nuclear Corp. (Boston, MA). Radioinert T and 5␣-dihydrotestosterone were supplied by Steraloids (Wilton, NH, USA). Sigma Chemical Co. (St. Louis, MO) supplied NADPH. Finasteride was obtained by extraction from Proscar (Merck, Sharp & Dohme). The tablets were crushed, extracted with chloroform and the solvent was eliminated in vacuum; the crude product was purified by silica gel column chromatography. 2.2. Synthesis of the steroidal derivatives 2.2.1. Synthesis of steroidal derivatives 5–11 2.2.1.1. 3β-Acetoxy-5α, 6α-epoxypregn-16-ene-20-one 5. A solution of steroid 4 (1 g, 2.82 mmol) and mchloroperbenzoic acid (1.62 g) in chloroform (50 ml) was stirred at room temperature for 30 min. Upon termination of the epoxidation, a saturated aqueous solution of sodium bicarbonate (84 ml) containing sodium bisulfite (1 g) was added. The reaction mixture was extracted three times with chloroform; the organic phase was washed with water, dried with sodium sulfate and the solvent was removed in vacuum. The crude product was recrystallized from methanol. Yield 1.01 g, 2.7 mmol (97%) of pure product 5, mp 170–172 ◦ C. UV (nm): 238 (ε = 10100). IR (KBr) cm−1 : 2943, 1734, 1665 and 1588. 1 H-NMR (CDCl ) δ: 0.81 (3Hs, H-18), 1.1 (3Hs, H-19), 2.0 3 (3Hs, H-21), 2.2 (3Hs, acetoxy), 2.9 (1Hd, J = 2 Hz, epoxy at C-6), 4.7 (1Hd, J = 2 Hz, H-3). 13 C-NMR (CDCl3 ) δ: 15.8 (C-18), 16.9 (C-19), 21.3 (C-21), 27.2 (acetoxy), 63.2 (C6), 65.3 (C-5), 144.1 (C-16), 155.2 (C-17), 170.5 (acetoxy carbonyl), 196.7 (C-20). MS m/z 372 (M+). 2.2.1.2. 3β-Acetoxy-5-hydroxypregn-16-ene-6,20-dione 6. To a solution of steroid 5 (1 g, 2.7 mmol) in acetone (50 ml) was added dropwise a solution of chromium trioxide (1.05 g, 10.5 mmol) in water (5 ml) at 0 ◦ C during 10 min. The re-
sulting mixture was allowed to warm up to room temperature and again the same amount of chromium trioxide was added in the same manner. The mixture was diluted with cold water (150 ml) and the precipitated product was filtered and dried. It was recrystallized from methanol; yield 852 mg, 2.19 mmol (81%) of pure product 6, mp 244–245 ◦ C. UV (nm) 238 (ε = 10200). IR (KBr) cm−1 : 3409, 2940, 1730, 1700, 1685 and 1600. 1 H-NMR (CDCl3 ) δ: 0.8 (3Hs, H18), 1.1 (3Hs, H19), 2.0 (3Hs, H-21), 2.2 (3Hs, acetoxy), 4.8 (1Hm, H-3), 6.6 (1Hq, J = 2 Hz, H-16). 13 C-NMR (CDCl3 ) δ: 15.8 (C-18), 17.0 (C-19), 22.0 (C-21), 28.0 (acetoxy), 80.0 (C5), 143.8 (C-16), 155.0 (C-17), 173.0 (acetoxy carbonyl), 197.2 (C-20), 212.8 (C-6). MS m/z 388 (M+). 2.2.1.3. 3β-Acetoxypregna-4,16-diene-6,20-dione 7. To a solution of steroid 6 (1 g, 2.57 mmol) in pyridine (10 ml) was added dropwise under a nitrogen atmosphere at 0 ◦ C thionyl chloride (1 ml). The resulting solution was stirred at room temperature for 45 min. Iced water (100 ml) was added and it was extracted three times with ethyl acetate. The organic phase was washed with 10% aqueous hydrochloric acid, 5% aqueous sodium bicarbonate and water. It was dried with sodium sulfate and the solvent was removed in vacuum. The crude product was recrystallized from ethyl acetate-hexane. Yield 623 mg, 1.69 mmol (66%) of pure product 7, mp 193–195 ◦ C. UV (nm): 239 (ε = 10300), 243 (ε = 6200). IR (KBr) cm−1 : 2942, 1735, 1691 and 1635. 1 H-NMR (CDCl3 ) δ: 0.9 (3Hs, H-18), 1.0 (3Hs, H-19), 2.1 (3Hs, H-21), 2.3 (3Hs, acetoxy), 5.1 (1Hm, H-3), 6.0 (1Hq, J = 2 Hz, H-4), 6.7 (1Hq, J = 2 Hz, H-16). 13 C-NMR (CDCl3 ) δ: 15.7 (C-18), 19.6 (C-19), 22.0 (C-21), 29.5 (acetoxy), 70.1 (C-3), 129.0 (C-4), 143.7 (C-16), 147.2 (C-5), 154.6 (C-17), 170.6 (acetoxy carbonyl), 195.0 (C-20), 201.7 (C-6). MS m/z 370 (M+). 2.2.1.4. 3β-Hydroxypregna-4,16-diene-6,20-dione 8. A solution of steroid 7 (1 g, 2.7 mmol), 2% aqueous sodium hydroxide solution (10 ml) in methanol (150 ml) was stirred at room temperature for 20 min. Upon termination of the hydrolysis, water (200 ml) was added and the precipitated product was filtered and dried. It was recrystallized from methanol. Yield 531 mg, 1.62 mmol (60%) of pure product 8, mp 168–170 ◦ C. UV (nm) 238 (ε = 10100), 242 (ε = 6100). IR (KBr) cm−1 : 3430, 2941, 1688, 1663 and 1630. 1 H-NMR (CDCl3 ) δ: 0.9 (3Hs, H-18), 1.0 (3Hs, H-19), 2.2 (3Hs, H-21), 4.3 (1Ht, J = 2 Hz, H-3), 6.2 (1Hq, J = 2, H-4), 6.7 (1Hq, J = 2, H-16). 13 C-NMR (CDCl3 ) δ: 15.7 (C-18), 19.8 (C-19), 27.1 (C-21), 67.1 (C-3), 133.2 (C-4), 143.9 (C-16), 146.5 (C-5), 154.8 (C-17), 196.6 (C-20), 202.4 (C-6). MS m/z 328 (M+). 2.2.1.5. Pregna-4,6-diene-3,6,20-trione 9. To a solution of steroid 8 (1 g, 2.57 mmol) in acetone (50 ml) was added dropwise a solution of chromium trioxide (1.05 g, 10.5 mmol) in water (5 ml) at 0 ◦ C during 10 min. The resulting mixture was allowed to warm to room temperature and again the same amount of chromium trioxide was added in the same manner. The reaction product was diluted with cold water (150 ml)
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and the precipitated product was filtered and dried. It was recrystallized from methanol. Yield 676 mg, 2.07 mmol (80%) or pure product 9, mp 207–209 ◦ C. UV (nm) 239 (ε = 10500), 252 (ε = 10800). IR (KBr): 2940, 1690, 1675 and 1600. 1 HNMR (CDCl3 ) δ: 0.9 (3Hs, H-18), 1.2 (3Hs, H-19), 2.1 (3Hs, H-21), 6.3 (1Ht, J = 2 Hz, H-4), 6.8 (1Ht, J = 2 Hz, H-16). 13 C-NMR (CDCl ) δ: 16.0 (C-18), 18.5 (C-19), 26.5 (C-21), 3 133.8 (C-4), 144.0 (C-16), 147.0 (C-5), 155.0 (C-17), 196.2 (C-20), 200.0 (C-3), 202.6 (C-6). MS m/z 326 (M+). 2.2.1.6. 3β-Benzoyloxypregna-4,16-diene-6,20-dione 10. A solution of steroid 8 (1 g, 3.04 mmol), benzoic acid (1 g, 8.1 mmol), dicyclohexylcarbodiimide (1 g, 5.0 mmol) and 4,4-dimethylaminopyridine (0.6 g, 4.9 mmol) in methylene chloride 50 ml was stirred at room temperature for 1.5 h. Upon completion of the esterification, the solution was poured in ice-water (150 ml) and extracted three times with chloroform; the organic phase was washed with water, dried with sodium sulfate and the solvent was removed in vacuum. The crude product was dissolved in ethyl acetate (30 ml) and filtered through a column containing aluminum oxide (3 g). The filtrate was concentrated and the desired ester 10 precipitated; yield 900 mg, 2.0 mmol (66%), mp 188–190 ◦ C. UV (nm) 239 (ε = 10300), 243 (ε = 6200). IR (KBr) cm−1 : 3085, 2943, 1723, 1690, 1665 and 1630. 1 H-NMR (CDCl ) δ: 0.9 (3Hs, H-18), 1.1 (3Hs, H-19), 2.3 3 (3Hs, H-21), 5.8 (1Ht, J = 2 Hz, H-3), 6.2 (1Hq, J = 2 Hz, H-4). 13 C-NMR (CDCl3 ) δ: 15.6 (C-18), 19.6 (C-19), 27.1 (C-21), 70.0 (C-3), 131.2 (C-4), 143.7 (C-16), 148.2 (C-5), 154.9 (C-17), 165.4 (ester carbonyl), 196.5 (C-20), 201.8 (C-6). MS m/z 432 (M+). 2.2.1.7. 3β-(p-Fluorobenzoyloxy)pregna-4,16-diene-6,20dione 11. A solution of steroid 8 (1 g, 3.04 mmol), p-fluorobenzoic acid (1 g, 7.14 mmol), dicyclohexylcarbodiimide (1 g, 5 mmol) and 4-dimethylaminopyridine (0.6 g, 4.9 mmol) in methylene chloride (100 ml) was stirred at room temperature for 1.5 h. Upon completion of the esterification, the solution was poured into iced water (150 ml) and extracted three times with chloroform; the organic phase was washed with water, dried with sodium sulfate and the solvent was removed in vacuum. The crude product was dissolved in ethyl acetate (30 ml) and filtered through a column containing aluminum oxide (3 g) to remove the dicyclohexylurea. The filtrate was concentrated and the desired ester 11 precipitated; yield 891 mg, 1.98 mmol (65%), mp 244–246 ◦ C. UV (nm) 238 (ε = 10100), 242 (ε = 6400). IR (KBr) cm−1 : 3075, 2952, 1730, 1680, 1670 and 1629. 1 H-NMR (CDCl3 ) δ: 0.8 (3Hs, H-18), 1.2 (3Hs, H-19), 2.2 (3Hs, H-21), 5.4 (1Ht, J = 2 Hz, H-3), 6.4 (1Hq, J = 2 Hz, H-4), 6.7 (1Ht, J = 2 Hz, H-16). 13 C-NMR (CDCl3 ) δ: 16.0 (C-18), 20.5 (C-19), 28.2 (C-21), 70.5 (C-3), 132.4 (C-4), 144.2 (C-16), 147.9 (C-5), 153.9 (C-17), 164.9 (ester carbonyl), 195.8 (C-20), 201.3 (C-6). MS m/z 450 (M+).
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2.3. Animals and tissues Adult male golden hamsters (150–200 g) were obtained from the Metropolitan University in Xochimilco, Mexico. Gonadectomies were performed under pentobarbital anesthesia 30 days prior to the experiments and the animals were sacrificed with CO2 . This protocol was approved by the Institutional Care and Use Committee of the Metropolitan University of Mexico (UAM). Human prostate from cadaver was kindly provided by Dr. Avissai Alc´antara at The General Hospital (SS) in Mexico City, and stored at −70 ◦ C. The prostate glands from hamsters were immediately removed, blotted and weighed prior to their use. Frozen human prostate was thawed on ice and minced with scissors. Unless specified, the following procedures were carried out at 4 ◦ C. The animal and human tissues were homogenized with a tissue homogenize (model 985–370; variable speed 5000–30,000 rpm, Biospec Products, Inc.). 2.4. In vitro experiments Tissues were homogenized in two volumes of medium A (20 mM sodium phosphate, pH 6.5 containing 0.32 M sucrose, 0.1 mM dithiothreitol Sigma-Aldrich, Inc.) with a tissue homogenizer. Homogenates were centrifuged at 1500 × g for 20 min [13] in a SW 60 Ti rotor (Beckman instruments, Palo Alto, CA). The pellets were separated, washed with three tissue volumes of medium A and centrifuged two additional times at 440 g at 0 ◦ C for 10 min [13]. The washed pellets were suspended in medium A and kept at −70 ◦ C. The suspension (6.8 mg protein/ml for hamster and 5 mg/ml for human prostates, determined by the Bradford method [14]) was used as source of 5␣-reductase. 2.5. Determination of 5α-reductase activities The enzyme 5␣-reductase was assayed as previously described [13]. The reaction mixture contained a final volume of 1 ml: 1 mM dithiothreitol, sodium phosphate buffer (40 mM, at pH 7 for hamster [15] and 6.5 for human prostates [13]), 2 mM, NADPH, 2 nM [1,2,6,7-3 H]T. The reaction in duplicate was started when it was added to the enzymatic fraction (134 g protein), incubated at 37 ◦ C for 60 min, and stopped by mixing with 1 ml of dichloromethane; this was considered as the end point. Incubation without tissue was used as control. The fraction of dichlorometane was separated and the extraction was repeated four more times. The extract was evaporated under a nitrogen stream to dryness and suspended on 50 l of methanol that was spotted on TLC Keiselgel 60 F254 plates. T and DHT were used as carriers and the plates were developed in chloroform:acetone = 9:1. The plates were airdried and the chromatography was repeated two more times. The T standard was visualized under UV lights (254 nm) and DHT was detected using phosphomolibdic acid reagent with a posterior heating of the plate. DHT develops a classical dark
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blue color. DHT containing areas were cut off and the strips were soaked in 5 ml in Ultima Gold (Packard) and the radioactivity was counted in the scintillation counter (Packard tri-carb 2100 TR). When the assay was repeated, we observed that the 5␣reductase enzyme from hamster and human prostate remained active for more than 4 months when it was stored at −70 ◦ C.
Fig. 1. Steroidal structures.
2.8. Toxicological assays 2.6. Determination of 50% inhibitory concentration of steroids 3, 5, 6, 7, 9, 10 and 11 in gonadectomized hamster and human prostatic 5α-reductase In order to calculate the IC50 (values the concentration of compounds 3, 5, 6, 7, 9, 10, and 11 required to inhibit 5␣-reductase activity by 50%), six series of tubes containing increasing concentrations of these steroids (10−11 –10−3 M) were incubated in duplicate, in the presence of: 1 mM of dithiothreitol, 40 mM sodium phosphate buffer pHs of 7 for hamster enzymatic fraction, or 6.5 for human; 2 mM NADPH, 2 nM [1,2,6,7-3 H]T and 134 g of protein from enzymatic fraction in a final volume of 1 ml. The reaction was carried out in duplicate at 37 ◦ C for 60 min adding 1 ml of dichloromethane to stop the reaction. The amount of DHT formed was determined as we detailed above. 2.7. In vivo experiments The effect of the new steroids 5, 6, 7, 9, 10 and 11 on the prostate of male hamsters, which had been gonadectomized 30 days prior to the experiment, was determined on nine groups of four animals per experiment, which were selected at random. The animals were kept in a room with controlled temperature (22 ◦ C) and periods of 12 h light:12 h dark. Food and water were provided ad libitum. For the daily subcutaneous injections, 400 g of the steroids 3, 5, 6, 7, 9, 10 and 11 were dissolved in 200 l of sesame oil and administered for 6 days together with 200 g of T. Three groups of animals were kept as control; one was injected with 200 l of sesame oil, the second one with 200 g of testosterone and the third one with T and finasteride for 6 days. After the treatment, the animals were sacrificed by CO2 and the prostate glands were dissected and weighed. Four separate experiments were performed for each group of steroid treated animals. The results (Table 2) were analyzed using one-way analysis of variance with EPISTAT software. In order to demonstrate that compound 5 is only an inhibitor of the enzyme 5␣-reductase and not an androgen antagonist, three groups of gonadectomized animals were used. The first served as a control, the second was stimulated with DHT and the third was treated with 200 g of DHT and 400 g of steroid 5. These experiments were carried out exactly in the same manner as those of T.
For the determination of toxicological effect of compound 5, we carried out two experiments. 2.8.1. Experiment 1 Determination of lethal concentration 50 in Artemia salina (brine shrimp larvae) assay [16]. The control in this experiment consisted of Artemia salina culture having 10 artemias each, incubated by triplicate in 2 ml of 10% tween in saline solution exposed to sun light and oxygen for 24 h. The experiment with compound 5 was carried out in the same manner in the presence of three different concentrations of the steroidal compound (10−6 –10−4 M). At the end of both experiments the number of alive and dead Artemias was counted; the results were plotted by the Redd-Muench method [17]. 2.8.2. Experiment 2 In vivo acute evaluation of lethal dose 50 in mice. For the determination of the lethal dose 50 in mice, we used four groups of six animals each. The male animals ICR, were provided by Harlan (Mexico) and weighed 25–30 g. The four groups were distributed in the following manner: (a) Intact control. (b) The animals were injected with sesame oil as vehicle. (c) Compound 5 was administrated subcutaneously in four different doses: 118.7, 237.5; 475 and 950 mg/kg [18–19]. (d) Finasteride was used as known control and was injected subcutaneously as described in (c). The behavior of each group of animals was observed at 1, 12, 24, 48 and 72 h prior to injections (Fig. 1).
3. Results 3.1. Synthesis of the steroidal derivatives Compounds 5–11 were prepared from the commercially available 16-dehydropregnenolone acetate 4 (Fig. 2). Compound 4 on treatment with m-chloroperbenzoic acid in chloroform afforded the 5,6-epoxy derivative 5. The hydroxycarbonyl derivative 6 was obtained when the epoxy compound 5 was treated with chromium trioxide in acetone–water. Elimination of the hydroxyl group in 6 with thionyl chloride,
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Fig. 2. Reaction sequence.
yielded the conjugated ketone 7; when this compound was hydrolyzed with sodium hydroxide in methanol, it afforded the alcohol 8. This compound was oxidized with chromium trioxide in acetone–water to the diene–trienone derivative 9. Esterification of the free hydroxyl group in 8 with benzoic acid and dicyclohexylcarbodiimide afforded the corresponding benzoate ester 10. The p-fluorobenzoate ester 11 was obtained when the alcohol 8 was esterified in the same manner with p-fluorobenzoic acid.
velopment of inhibition plots. Unmodified [3 H]T was identified from control incubations which did not contain tissue. 3.3. Determination of 50% inhibitory concentration of the novel compounds in hamster prostate The concentrations of compounds 3, 5, 6, 7, 9, 10 and 11 required for inhibiting 5␣-reductase activity by 50% (IC50 ) were determined from the inhibition curves using different concentrations of the steroids and are shown in Table 1.
3.2. In vitro experiments The in vitro biological activity of compounds 3, 5, 6, 7, 9, 10 and 11 was determined in gonadectomized hamster and human prostate glands homogenized and centrifuged to obtain the prostatic enzyme. The activity of hamster and human 5␣-reductases was assessed incubating the enzymatic fractions with 2 nM [3 H]T. The dichloromethane extracts from castrated male hamster and human prostates were subjected to TLC analysis. The zone corresponding to DHT standard (Rf value of 0.67) of the experimental chromatogram was cut off, soaked in Ultima Gold and the radioactivity determined. This result was considered to be 100% of the activity of 5␣-reductase for de-
Table 1 The IC50 values were determined for finasteride and the synthesized steroids 5, 6, 7, 9, 10, 11 with human and gonadectomized hamsters prostatic 5␣reductase; they represent the concentration of the steroid that inhibits 50% of 5␣-reductase activity and were determined as described in Section 2 Treatment
Hamster prostatic enzyme IC50 (M)
Human prostatic enzyme IC50 (M)
T+3 T+5 T+6 T+7 T+9 T + 10 T + 11
1.0 × 10−8 6.5 × 10−11 1.0 × 10−4 6.0 × 10−11 9.2 × 10−10 6.0 × 10−11 2.0 × 10−4
8.5 × 10−9 6.3 × 10−11 2.0 × 10−4 6.5 × 10−11 8.5 × 10−10 7.0 × 10−11 3.5.0 × 10−4
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The IC50 values obtained from hamster and human prostate’s 5␣-reductase enzyme for compounds 5, 7, 9 and 10, were similar; however they were much lower than that for finasteride 3, thus indicating a much higher 5␣-reductase inhibitory activity (Table 1). Since the weight of the prostate gland depends on the 5␣reduced androgens [20], it was important to determine the effect of the new steroidal compounds 5, 6, 7, 9, 10 and 11 on the activity of 5␣-reductase enzyme. 3.4. In vivo experiments After castration, the weight of the male hamster prostates decreased (p < 0.005) compared to that of the normal glands. Treatment with vehicle alone did not change this condition, whereas s.c. injections of 200 g of T for 6 days significantly increased (p < 0.005) the weight of the prostate glands in castrated male hamsters (Table 2). When testosterone (T) and finasteride were injected together, the weight of the prostate decreased (p < 0.005) as compared to testosterone-treated animals (Table 2). However, when the same experiment was carried out with compounds, 5, 6, 7, 9, 10 and 11 the weight of the prostate decreased significantly (P < 0.005) only in the case of steroid 5 thus showing a comparable anti-androgenic effect to that of finasteride 3 (Table 2). The experiment with DHT showed that compound 5 is only an inhibitor of the 5␣-reductase and not an antagonist to the androgen receptor. This fact is demonstrated by the prostate gland weights such as: control 39.1 ± 8.5 mg: DHTstimulated weight 110.7 mg, and compound 5 plus DHT experiment of 101.7 ± 16.5 mg. 3.5. Toxicological assay The lethal dose 50 was determined in Artemia salina model. The results from Experiment 1, indicated that in the control experiment, all of the Artemias remained alive, whereas in Table 2 Weight of prostate glands ±standard deviations from animals receiving different s.c. treatments for 6 days Treatment
Prostate weight (mg)
Control T T+3 T+5 T+6 T+7 T+9 T + 10 T + 11
48.5 87.6 55.0 57.8 78.7 77.1 74.1 76.0 75.0
± ± ± ± ± ± ± ± ±
9.59 10.2 10.7 4.8 14.8 15.6 15.9 18.6 15.0
Significant differences (p < 0.05) between T and control (vehicle) and T plus finasteride or T plus 5, were observed. Three groups of animals were kept as control; one was injected with 200 l of sesame oil (Vehicle), the second one with 200 g of T and the third one T with plus finasteride 3 for 6 days (see Section 2).
Fig. 3. Determination of lethal doses 50 (LC50) by Redd-Muench plot. Cultures of Artemia salina were kept in the presence of the vehicle and compound 5. The two plots (dead and survivors) join in the LC50 point, and represents the doses required for the Artemia salina population.
cultures containing different concentrations of compound 5 the results indicated that the low and high concentrations exhibited the highest number of survivors. These data show very clearly that the epoxy compound does not exhibit any toxicological effects, since most of the crustacean remained alive. An increase of the concentration of compound 5 in the culture medium produced some dead crustacea (Fig. 3). This experiment shows very clearly that only very elevated concentrations of compound 5 resulted in the death of some crustacea. 3.5.1. In vivo acute evaluation of lethal dose 50 in mice No change was observed in the intact as well as in the vehicle controls. Compound 5 as well as finasteride did not produce any lethal and toxicological effects at the doses used. 4. Discussion In this paper we assessed the 5␣-reductase inhibitory activity of six new pregnane derivatives 5, 6, 7, 9, 10 and 11 by comparing their IC50 values. The results from this study with hamster and human prostate enzymes showed very clearly that compounds 5, 7, 9 and 10 (Table 1) are much better inhibitors for the enzyme 5␣-reductase than the presently used Proscar 3 (finasteride). These results confirm also the similarity of both hamster and human 5␣-reductase enzymes. The fact that the optimum pH for hamster’s 5␣-reductase activity at 2 nM is 7 [13] whereas that for human is 6.5 at the same concentration indicates very clearly that some differences in their activity are present. Although the active steroidal derivatives 5, 7, 9 and 10 have different functional groups, they all have one moiety in common, the ␣,-unsaturated C-20-carbonyl group. Accessible electrophilic -carbon of an ␣,-unsaturated carbonyl moiety reacts very readily with a variety of nu-
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cleophiles to form Michael aducts. Several years ago, we postulated that the inhibition of the enzyme 5␣-reductase is based on the Michael type addition reaction of the enzyme 5␣-reductase to the steroidal enone or dienone [12,21]. The results from this study indicated that the first step in the inhibition of the enzyme 5␣-reductase consists in the formation of an enzyme-steroid activated complex. In a subsequent step, the nucleophilic portion of the enzyme (amino group) attacks the conjugated double bond of the steroid in a Michael type addition reaction to form an irreversible adducts. This concept explains very well the high 5␣-reductase inhibitory activity of the steroidal derivatives 5, 7, 9 and 10 which are Michael acceptors (Table 1). In steroid 5, (epoxy compound) the nucleophilic enzyme attacks the electrophilic center at C-6 and this process inhibits the enzyme. Although the chemical structure of the compounds with low activity (6 and 11) is very similar to that of the active steroids 5, 7, 9 and 10, apparently the presence of a fluorine atom in 11 and a hydroxyl group in compound 6 reduces the solubility of these steroidal derivatives and thus diminishes the effectiveness of the Michael type addition reaction. This mechanism has recently been confirmed for the inhibition of the enzyme ubiquitin isopeptidase by prostaglandins containing the cyclopentenone moiety [22]. In vivo experiments demonstrated that only steroid 5 (weight of the prostate gland 57.8 mg) has a comparable activity to the commercially available finasteride 3 (weight of the prostate gland 55.0 mg) (Table 2). All other compounds 6, 7, 9, 10 and 11 showed a weak activity as compared to testosterone treated animals. The in vivo activity of steroid 5 in blocking T-induced prostate weight gain is not due to the antagonist effect on the androgen receptor, since this steroid did not block the DHT-induced prostate weight gain. In order to develop a good inhibitor for the enzyme 5␣reductase which could be used for the treatment of human androgen-dependent disorders such as: benign prostatic hyperplasia and prostate cancer, it is imperative to carry out preclinical evaluation with the synthesized compounds 5, 6, 7, 9, 10 and 11 in vitro and also with animals. The data from our studies indicate that the in vivo efficiency of the 5␣-reductase inhibitors may depend not only on its affinity for the enzyme but also on the rate and extend of absorption, as well as the half life of the parent compound and active metabolites in the prostate. Our studies have also provided evidence about the similarity between hamster and human 5␣-reductase enzyme behavior in the presence of inhibitors at their corresponding pH and concentration optimum. The result of the toxicological assays carried out with Artemia salina cultures showed very clearly that steroid 5 did not produce any toxicological and lethal effects, and as a consequence of this it could be considered a save compound with high pharmacological potential. Since compound 5 is in human prostate enzyme about 100 times more active as 5␣-reductase inhibitor than the commercially available Proscar 3 (finasteride) (Table 1) and does
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not produce any toxicological effects in the near future, this compound will be evaluated on a clinical level. The synthesis of this compound is very simple and much cheaper than that of finasteride 3, and this steroidal derivative could represent a magnificent alternative for the treatment of androgendependent diseases.
Acknowledgements The authors gratefully acknowledge the financial support of CONACYT and DGAPA for projects 33450-M and 200301. The authors would like to thank Alejandro OrtizOsornio and Ruth Bustamante-Garc´ıa for technical assistance in toxicological assays.
References [1] Li X, Chen C, Singh SM, Labrie F. The enzyme and inhibitors of 4-ene-3-oxosteroid 5␣-oxidoreductase. Steroids 1995;60:430–41. [2] Geller J, Albert J, Lopez D, Geller S, Niwayama G. Comparison of androgen metabolites in benign prostatic hypertrophy (BPH) and normal prostate. J Clin Endocrinol Metab 1976;43:486–8. [3] Sitteri PK, Wilson JD, Mayfield JA. Dihydrotestosterone in prostatic hypertrophy I. The formation and content of dihydrotestosterone in the hypertrophic prostate of man. J Clin Invest 1970;49:1737–45. [4] Russell WD, Wilson JD. Steroid 5-alpha reductase: two genes/two enzymes. Annu Rev Biochem 1994;63:25–61. [5] Brooks JR, Harris GS, Sandler M, Smither HJ, editors. Design of enzyme inhibitor as drug, vol. 2. Oxford University Press; 1984. p. 495–542. [6] Mc Connel J, Wilson JD, George FG, Geller J, Pappas F, Stoner E. Finasteride, an inhibitor of 5-alpha reductase, suppresses prostatic dihydrotestosterone in men with benign prostatic hyperplasia. J Clin Endocrinol Metab 1992;74:505–9. [7] Machetti F, Guarna A. Novel inhibitors of 5␣-reductase. Expert Opin Ther Patents 2002;12:201–15. [8] Gormley GJ, Stoner E, Bruskewiz RC, Imperato-McGinley J, Walsh PC, McConnel JD, et al. The effect of finasteride in men with benign prostatic hyperplasia. N Engl J Med 1992;327:1185–91. [9] Cabeza M, Heuze I, Bratoeff E, Murillo E, Ram´ırez E, Lira A. New progesterona esters as 5␣-reductase inhibitors. Chem Pharm Bull 2001;49:1081–4. [10] Flores E, Cabeza M, Quiroz A, Bratoeff E, Garc´ıa G, Ram´ırez E. Effect of a novel steroid (PM-9) on the inhibition of 5␣-reductase present in Penicillium crustosum broths. Steroids 2003;68:271–5. [11] Bratoeff E, Ram´ırez E, Flores E, Valencia N, S´anchez M, Heuze I, et al. Molecular interactions of new pregnendione derivatives. Chem Pharm Bull 2003;51:1132–6. [12] Flores E, Bratoeff E, Cabeza M, Ram´ırez E, Quiroz A, Heuze I. Steroid. 5␣-Reductase inhibitors. Mini Rev Med Chem 2003;3:225–37. [13] Hirosumi J, Nakayama O, Fagan T, Sawada K, Chida N, Inami M, et al. FK143, a novel nonsteroidal inhibiton of steroid 5␣-reductase: (1) in vitro effects on human and animal prostatic enzymes. J Steroid Biochem Mol Biol 1995;52:357–63. [14] Bradford MM. A rapid and sensitive method for the quantization of micrograms quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1986;72:248–54. [15] Wright SA, Douglas RC, Thomas LN, Lazier CB, Rittmaster RS. Androgen-induced regrowth in the castrated rat ventral prostate: role of 5␣-reductase. Endocrinology 1999;140:4509–15.
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[16] Lewan L, Andersson M, Morales-G´omez P. The use of Artemia salina in toxicity testing. ATLA 1992;20:297–301. [17] Sam TW. Toxicity testing using the brine shrimp Artemia salina. In: Colegate SM, Mulgneux RJ, editors. Bioactive natural products. Detection, isolation, and structural determination. United States of America: CRC Press (ed), Inc.; 1993. p. 441–55. [18] Hilleshein HG, Hoffman H. Data on acute toxicity of the progestins STS 557. Exp Clin Endocrinol 1983;81:175–8. [19] Lorke D. A new approach to practical acute toxicity testing. Arch Toxicol 1993;54:275–87.
[20] Cabeza M, Bratoeff E, Heuze I, Ram´ırez E, S´anchez M, Flores E. Enzymatic and binding activities of 5␣-reductase and androgen receptors in hamster prostate. Proc West Pharmacol Soc 2003;46:128– 31. [21] Bratoeff E, Rubio M, Ram´ırez E, Garc´ıa J. Pharmacological and theoretical evaluation of new antindogens. Chem Today 1998;16:33– 5. [22] Verbitski S, Mullally J, Fitzpatrick F, Ireland C. Punaglandins, chlorinated prostaglandins, function as potent Michael receptors to inhibit ubiquitin isopeptidase activity. J Med Chem 2004;47:2062–70.
New 5␣-reductase inhibitors: In vitro and in vivo effects V´ıctor P´erez-Ornelas a , Marisa Cabeza b, ∗ , Eugene Bratoeff a , Ivonne Heuze b , Mauricio S´anchez b , Elena Ram´ırez a , Elia Naranjo-Rodr´ıguez a b
a Department of Pharmacy, Faculty of Chemistry, National University of Mexico City, Mexico D.F., Mexico Departments of Biological Systems and Animal Production of Metropolitan University of M´exico Center Xochimilco, Calzada Del Hueso 1100, 04960 Mexico D.F., Mexico
Received 19 May 2004; received in revised form 13 October 2004; accepted 5 November 2004
Abstract The enzyme 5␣-reductase is responsible for the conversion of testosterone (T) to its more potent androgen dihydrotestosterone (DHT). This steroid had been implicated in androgen-dependent diseases such as: benign prostatic hyperplasia, prostate cancer, acne and androgenic alopecia. The inhibition of 5␣-reductase enzyme offers a potentially useful treatment for these diseases. In this study, we report the synthesis and pharmacological evaluation of several new 3-substituted pregna-4, 16-diene-6, 20-dione derivatives. These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological activity of the new steroidal derivatives was determined in vivo as well as in vitro experiments. In vivo experiments, the anti-androgenic effect of the steroids was demonstrated by the decrease of the weight of the prostate gland of gonadectomized hamster treated with T plus finasteride or the new steroids. The IC50 value of these steroids was determined by measuring the conversion of radio labeled T to DHT. The results of this study carried out with 5␣-reductase enzyme from hamster and human prostate showed that four of the six steroidal derivatives (5, 7, 9, 10) exhibited much higher 5␣-reductase inhibitory activity, as indicated by the IC50 values than the presently used Proscar 3 (finasteride). The comparison of the weight of the hamster’s prostate gland indicated that compound 5 had a comparable weight decrease as finasteride. The overall data of this study showed very clearly those compounds 5, 7, 9, 10 are good inhibitors for the 5␣-reductase enzyme. © 2005 Elsevier Inc. All rights reserved. Keywords: 5␣-Reductase; 5␣-Reductase inhibitors; Prostate; Pregnadiene derivatives; In vivo effect of 5␣-reductase inhibitors; Epoxide toxicity
1. Introduction The normal activity of the NADPH-dependent 5␣reductase enzyme (EC 1.3.99.5) maintains testosterone’s 1 (T) biological functions: anabolic actions and spermatogenesis of humans as well as the dihydrotestosterone 2 mediated effects such as, increased facial and body hair, acne, scalp hair recession, and prostate enlargement [1]. Abnormally high 5␣-reductase activity in humans results in excessively high DHT levels in peripheral tissues, which is implicated in the pathogenesis of prostate cancer, benign prostatic hyperplasia ∗
Corresponding author. Tel.: +52 55 5483 72 60; fax: +52 55 5483 72 60. E-mail address: [email protected] (M. Cabeza).
0039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2004.11.008
(BPH), acne, and male patter baldness [2,3], thus suggesting that both the enzyme 5␣-reductase and DHT play important physiological and pathological roles in humans. Therefore, the suppression of androgen action by 5␣-reductase inhibitors is a logical treatment for 5␣-reductase activity disorders. Furthermore, since the beginning of the last decade, two types of 5␣-reductase enzyme had been identified 1 and 2 [4,5]; the identification of these two isozymes opened the door for specific and selective inhibition. The most extensively studied class of 5␣-reductase inhibitors are the 4-azasteroids [6,7], which include the drug finasteride 3. This compound is the first 5␣-reductase inhibitor approved in the USA for the treatment of BPH. This drug has approximately a 100-fold greater affinity for type
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2–5␣-reductase, than for the type 1 enzyme. In humans, finasteride decreases prostatic DHT levels by 70–90% and reduces prostate size, while T tissue levels remain constant [8]. The use of finasteride demonstrated a sustained improvement in the treatment of androgen-dependent diseases and it also reduces the prostate specific antigen (PSA) levels [8]. Recently, our group synthesized several new progesterone derivatives that considerably decreased the prostate growth produced by T [9–12]. Since these compounds showed a high biological activity [9–12], in this paper, we describe the synthesis and pharmacological evaluation of six similar compounds (5–11) based on the progesterone skeleton as inhibitors of hamster and human 5␣-reductase enzyme.
2. Experimental 2.1. Chemical and radioactive material Solvents were laboratory grade or better. (1, 2, 6, 7–3 H) Testosterone [3 H] T specific activity: 95 Ci/mmol was provided by New England Nuclear Corp. (Boston, MA). Radioinert T and 5␣-dihydrotestosterone were supplied by Steraloids (Wilton, NH, USA). Sigma Chemical Co. (St. Louis, MO) supplied NADPH. Finasteride was obtained by extraction from Proscar (Merck, Sharp & Dohme). The tablets were crushed, extracted with chloroform and the solvent was eliminated in vacuum; the crude product was purified by silica gel column chromatography. 2.2. Synthesis of the steroidal derivatives 2.2.1. Synthesis of steroidal derivatives 5–11 2.2.1.1. 3β-Acetoxy-5α, 6α-epoxypregn-16-ene-20-one 5. A solution of steroid 4 (1 g, 2.82 mmol) and mchloroperbenzoic acid (1.62 g) in chloroform (50 ml) was stirred at room temperature for 30 min. Upon termination of the epoxidation, a saturated aqueous solution of sodium bicarbonate (84 ml) containing sodium bisulfite (1 g) was added. The reaction mixture was extracted three times with chloroform; the organic phase was washed with water, dried with sodium sulfate and the solvent was removed in vacuum. The crude product was recrystallized from methanol. Yield 1.01 g, 2.7 mmol (97%) of pure product 5, mp 170–172 ◦ C. UV (nm): 238 (ε = 10100). IR (KBr) cm−1 : 2943, 1734, 1665 and 1588. 1 H-NMR (CDCl ) δ: 0.81 (3Hs, H-18), 1.1 (3Hs, H-19), 2.0 3 (3Hs, H-21), 2.2 (3Hs, acetoxy), 2.9 (1Hd, J = 2 Hz, epoxy at C-6), 4.7 (1Hd, J = 2 Hz, H-3). 13 C-NMR (CDCl3 ) δ: 15.8 (C-18), 16.9 (C-19), 21.3 (C-21), 27.2 (acetoxy), 63.2 (C6), 65.3 (C-5), 144.1 (C-16), 155.2 (C-17), 170.5 (acetoxy carbonyl), 196.7 (C-20). MS m/z 372 (M+). 2.2.1.2. 3β-Acetoxy-5-hydroxypregn-16-ene-6,20-dione 6. To a solution of steroid 5 (1 g, 2.7 mmol) in acetone (50 ml) was added dropwise a solution of chromium trioxide (1.05 g, 10.5 mmol) in water (5 ml) at 0 ◦ C during 10 min. The re-
sulting mixture was allowed to warm up to room temperature and again the same amount of chromium trioxide was added in the same manner. The mixture was diluted with cold water (150 ml) and the precipitated product was filtered and dried. It was recrystallized from methanol; yield 852 mg, 2.19 mmol (81%) of pure product 6, mp 244–245 ◦ C. UV (nm) 238 (ε = 10200). IR (KBr) cm−1 : 3409, 2940, 1730, 1700, 1685 and 1600. 1 H-NMR (CDCl3 ) δ: 0.8 (3Hs, H18), 1.1 (3Hs, H19), 2.0 (3Hs, H-21), 2.2 (3Hs, acetoxy), 4.8 (1Hm, H-3), 6.6 (1Hq, J = 2 Hz, H-16). 13 C-NMR (CDCl3 ) δ: 15.8 (C-18), 17.0 (C-19), 22.0 (C-21), 28.0 (acetoxy), 80.0 (C5), 143.8 (C-16), 155.0 (C-17), 173.0 (acetoxy carbonyl), 197.2 (C-20), 212.8 (C-6). MS m/z 388 (M+). 2.2.1.3. 3β-Acetoxypregna-4,16-diene-6,20-dione 7. To a solution of steroid 6 (1 g, 2.57 mmol) in pyridine (10 ml) was added dropwise under a nitrogen atmosphere at 0 ◦ C thionyl chloride (1 ml). The resulting solution was stirred at room temperature for 45 min. Iced water (100 ml) was added and it was extracted three times with ethyl acetate. The organic phase was washed with 10% aqueous hydrochloric acid, 5% aqueous sodium bicarbonate and water. It was dried with sodium sulfate and the solvent was removed in vacuum. The crude product was recrystallized from ethyl acetate-hexane. Yield 623 mg, 1.69 mmol (66%) of pure product 7, mp 193–195 ◦ C. UV (nm): 239 (ε = 10300), 243 (ε = 6200). IR (KBr) cm−1 : 2942, 1735, 1691 and 1635. 1 H-NMR (CDCl3 ) δ: 0.9 (3Hs, H-18), 1.0 (3Hs, H-19), 2.1 (3Hs, H-21), 2.3 (3Hs, acetoxy), 5.1 (1Hm, H-3), 6.0 (1Hq, J = 2 Hz, H-4), 6.7 (1Hq, J = 2 Hz, H-16). 13 C-NMR (CDCl3 ) δ: 15.7 (C-18), 19.6 (C-19), 22.0 (C-21), 29.5 (acetoxy), 70.1 (C-3), 129.0 (C-4), 143.7 (C-16), 147.2 (C-5), 154.6 (C-17), 170.6 (acetoxy carbonyl), 195.0 (C-20), 201.7 (C-6). MS m/z 370 (M+). 2.2.1.4. 3β-Hydroxypregna-4,16-diene-6,20-dione 8. A solution of steroid 7 (1 g, 2.7 mmol), 2% aqueous sodium hydroxide solution (10 ml) in methanol (150 ml) was stirred at room temperature for 20 min. Upon termination of the hydrolysis, water (200 ml) was added and the precipitated product was filtered and dried. It was recrystallized from methanol. Yield 531 mg, 1.62 mmol (60%) of pure product 8, mp 168–170 ◦ C. UV (nm) 238 (ε = 10100), 242 (ε = 6100). IR (KBr) cm−1 : 3430, 2941, 1688, 1663 and 1630. 1 H-NMR (CDCl3 ) δ: 0.9 (3Hs, H-18), 1.0 (3Hs, H-19), 2.2 (3Hs, H-21), 4.3 (1Ht, J = 2 Hz, H-3), 6.2 (1Hq, J = 2, H-4), 6.7 (1Hq, J = 2, H-16). 13 C-NMR (CDCl3 ) δ: 15.7 (C-18), 19.8 (C-19), 27.1 (C-21), 67.1 (C-3), 133.2 (C-4), 143.9 (C-16), 146.5 (C-5), 154.8 (C-17), 196.6 (C-20), 202.4 (C-6). MS m/z 328 (M+). 2.2.1.5. Pregna-4,6-diene-3,6,20-trione 9. To a solution of steroid 8 (1 g, 2.57 mmol) in acetone (50 ml) was added dropwise a solution of chromium trioxide (1.05 g, 10.5 mmol) in water (5 ml) at 0 ◦ C during 10 min. The resulting mixture was allowed to warm to room temperature and again the same amount of chromium trioxide was added in the same manner. The reaction product was diluted with cold water (150 ml)
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and the precipitated product was filtered and dried. It was recrystallized from methanol. Yield 676 mg, 2.07 mmol (80%) or pure product 9, mp 207–209 ◦ C. UV (nm) 239 (ε = 10500), 252 (ε = 10800). IR (KBr): 2940, 1690, 1675 and 1600. 1 HNMR (CDCl3 ) δ: 0.9 (3Hs, H-18), 1.2 (3Hs, H-19), 2.1 (3Hs, H-21), 6.3 (1Ht, J = 2 Hz, H-4), 6.8 (1Ht, J = 2 Hz, H-16). 13 C-NMR (CDCl ) δ: 16.0 (C-18), 18.5 (C-19), 26.5 (C-21), 3 133.8 (C-4), 144.0 (C-16), 147.0 (C-5), 155.0 (C-17), 196.2 (C-20), 200.0 (C-3), 202.6 (C-6). MS m/z 326 (M+). 2.2.1.6. 3β-Benzoyloxypregna-4,16-diene-6,20-dione 10. A solution of steroid 8 (1 g, 3.04 mmol), benzoic acid (1 g, 8.1 mmol), dicyclohexylcarbodiimide (1 g, 5.0 mmol) and 4,4-dimethylaminopyridine (0.6 g, 4.9 mmol) in methylene chloride 50 ml was stirred at room temperature for 1.5 h. Upon completion of the esterification, the solution was poured in ice-water (150 ml) and extracted three times with chloroform; the organic phase was washed with water, dried with sodium sulfate and the solvent was removed in vacuum. The crude product was dissolved in ethyl acetate (30 ml) and filtered through a column containing aluminum oxide (3 g). The filtrate was concentrated and the desired ester 10 precipitated; yield 900 mg, 2.0 mmol (66%), mp 188–190 ◦ C. UV (nm) 239 (ε = 10300), 243 (ε = 6200). IR (KBr) cm−1 : 3085, 2943, 1723, 1690, 1665 and 1630. 1 H-NMR (CDCl ) δ: 0.9 (3Hs, H-18), 1.1 (3Hs, H-19), 2.3 3 (3Hs, H-21), 5.8 (1Ht, J = 2 Hz, H-3), 6.2 (1Hq, J = 2 Hz, H-4). 13 C-NMR (CDCl3 ) δ: 15.6 (C-18), 19.6 (C-19), 27.1 (C-21), 70.0 (C-3), 131.2 (C-4), 143.7 (C-16), 148.2 (C-5), 154.9 (C-17), 165.4 (ester carbonyl), 196.5 (C-20), 201.8 (C-6). MS m/z 432 (M+). 2.2.1.7. 3β-(p-Fluorobenzoyloxy)pregna-4,16-diene-6,20dione 11. A solution of steroid 8 (1 g, 3.04 mmol), p-fluorobenzoic acid (1 g, 7.14 mmol), dicyclohexylcarbodiimide (1 g, 5 mmol) and 4-dimethylaminopyridine (0.6 g, 4.9 mmol) in methylene chloride (100 ml) was stirred at room temperature for 1.5 h. Upon completion of the esterification, the solution was poured into iced water (150 ml) and extracted three times with chloroform; the organic phase was washed with water, dried with sodium sulfate and the solvent was removed in vacuum. The crude product was dissolved in ethyl acetate (30 ml) and filtered through a column containing aluminum oxide (3 g) to remove the dicyclohexylurea. The filtrate was concentrated and the desired ester 11 precipitated; yield 891 mg, 1.98 mmol (65%), mp 244–246 ◦ C. UV (nm) 238 (ε = 10100), 242 (ε = 6400). IR (KBr) cm−1 : 3075, 2952, 1730, 1680, 1670 and 1629. 1 H-NMR (CDCl3 ) δ: 0.8 (3Hs, H-18), 1.2 (3Hs, H-19), 2.2 (3Hs, H-21), 5.4 (1Ht, J = 2 Hz, H-3), 6.4 (1Hq, J = 2 Hz, H-4), 6.7 (1Ht, J = 2 Hz, H-16). 13 C-NMR (CDCl3 ) δ: 16.0 (C-18), 20.5 (C-19), 28.2 (C-21), 70.5 (C-3), 132.4 (C-4), 144.2 (C-16), 147.9 (C-5), 153.9 (C-17), 164.9 (ester carbonyl), 195.8 (C-20), 201.3 (C-6). MS m/z 450 (M+).
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2.3. Animals and tissues Adult male golden hamsters (150–200 g) were obtained from the Metropolitan University in Xochimilco, Mexico. Gonadectomies were performed under pentobarbital anesthesia 30 days prior to the experiments and the animals were sacrificed with CO2 . This protocol was approved by the Institutional Care and Use Committee of the Metropolitan University of Mexico (UAM). Human prostate from cadaver was kindly provided by Dr. Avissai Alc´antara at The General Hospital (SS) in Mexico City, and stored at −70 ◦ C. The prostate glands from hamsters were immediately removed, blotted and weighed prior to their use. Frozen human prostate was thawed on ice and minced with scissors. Unless specified, the following procedures were carried out at 4 ◦ C. The animal and human tissues were homogenized with a tissue homogenize (model 985–370; variable speed 5000–30,000 rpm, Biospec Products, Inc.). 2.4. In vitro experiments Tissues were homogenized in two volumes of medium A (20 mM sodium phosphate, pH 6.5 containing 0.32 M sucrose, 0.1 mM dithiothreitol Sigma-Aldrich, Inc.) with a tissue homogenizer. Homogenates were centrifuged at 1500 × g for 20 min [13] in a SW 60 Ti rotor (Beckman instruments, Palo Alto, CA). The pellets were separated, washed with three tissue volumes of medium A and centrifuged two additional times at 440 g at 0 ◦ C for 10 min [13]. The washed pellets were suspended in medium A and kept at −70 ◦ C. The suspension (6.8 mg protein/ml for hamster and 5 mg/ml for human prostates, determined by the Bradford method [14]) was used as source of 5␣-reductase. 2.5. Determination of 5α-reductase activities The enzyme 5␣-reductase was assayed as previously described [13]. The reaction mixture contained a final volume of 1 ml: 1 mM dithiothreitol, sodium phosphate buffer (40 mM, at pH 7 for hamster [15] and 6.5 for human prostates [13]), 2 mM, NADPH, 2 nM [1,2,6,7-3 H]T. The reaction in duplicate was started when it was added to the enzymatic fraction (134 g protein), incubated at 37 ◦ C for 60 min, and stopped by mixing with 1 ml of dichloromethane; this was considered as the end point. Incubation without tissue was used as control. The fraction of dichlorometane was separated and the extraction was repeated four more times. The extract was evaporated under a nitrogen stream to dryness and suspended on 50 l of methanol that was spotted on TLC Keiselgel 60 F254 plates. T and DHT were used as carriers and the plates were developed in chloroform:acetone = 9:1. The plates were airdried and the chromatography was repeated two more times. The T standard was visualized under UV lights (254 nm) and DHT was detected using phosphomolibdic acid reagent with a posterior heating of the plate. DHT develops a classical dark
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blue color. DHT containing areas were cut off and the strips were soaked in 5 ml in Ultima Gold (Packard) and the radioactivity was counted in the scintillation counter (Packard tri-carb 2100 TR). When the assay was repeated, we observed that the 5␣reductase enzyme from hamster and human prostate remained active for more than 4 months when it was stored at −70 ◦ C.
Fig. 1. Steroidal structures.
2.8. Toxicological assays 2.6. Determination of 50% inhibitory concentration of steroids 3, 5, 6, 7, 9, 10 and 11 in gonadectomized hamster and human prostatic 5α-reductase In order to calculate the IC50 (values the concentration of compounds 3, 5, 6, 7, 9, 10, and 11 required to inhibit 5␣-reductase activity by 50%), six series of tubes containing increasing concentrations of these steroids (10−11 –10−3 M) were incubated in duplicate, in the presence of: 1 mM of dithiothreitol, 40 mM sodium phosphate buffer pHs of 7 for hamster enzymatic fraction, or 6.5 for human; 2 mM NADPH, 2 nM [1,2,6,7-3 H]T and 134 g of protein from enzymatic fraction in a final volume of 1 ml. The reaction was carried out in duplicate at 37 ◦ C for 60 min adding 1 ml of dichloromethane to stop the reaction. The amount of DHT formed was determined as we detailed above. 2.7. In vivo experiments The effect of the new steroids 5, 6, 7, 9, 10 and 11 on the prostate of male hamsters, which had been gonadectomized 30 days prior to the experiment, was determined on nine groups of four animals per experiment, which were selected at random. The animals were kept in a room with controlled temperature (22 ◦ C) and periods of 12 h light:12 h dark. Food and water were provided ad libitum. For the daily subcutaneous injections, 400 g of the steroids 3, 5, 6, 7, 9, 10 and 11 were dissolved in 200 l of sesame oil and administered for 6 days together with 200 g of T. Three groups of animals were kept as control; one was injected with 200 l of sesame oil, the second one with 200 g of testosterone and the third one with T and finasteride for 6 days. After the treatment, the animals were sacrificed by CO2 and the prostate glands were dissected and weighed. Four separate experiments were performed for each group of steroid treated animals. The results (Table 2) were analyzed using one-way analysis of variance with EPISTAT software. In order to demonstrate that compound 5 is only an inhibitor of the enzyme 5␣-reductase and not an androgen antagonist, three groups of gonadectomized animals were used. The first served as a control, the second was stimulated with DHT and the third was treated with 200 g of DHT and 400 g of steroid 5. These experiments were carried out exactly in the same manner as those of T.
For the determination of toxicological effect of compound 5, we carried out two experiments. 2.8.1. Experiment 1 Determination of lethal concentration 50 in Artemia salina (brine shrimp larvae) assay [16]. The control in this experiment consisted of Artemia salina culture having 10 artemias each, incubated by triplicate in 2 ml of 10% tween in saline solution exposed to sun light and oxygen for 24 h. The experiment with compound 5 was carried out in the same manner in the presence of three different concentrations of the steroidal compound (10−6 –10−4 M). At the end of both experiments the number of alive and dead Artemias was counted; the results were plotted by the Redd-Muench method [17]. 2.8.2. Experiment 2 In vivo acute evaluation of lethal dose 50 in mice. For the determination of the lethal dose 50 in mice, we used four groups of six animals each. The male animals ICR, were provided by Harlan (Mexico) and weighed 25–30 g. The four groups were distributed in the following manner: (a) Intact control. (b) The animals were injected with sesame oil as vehicle. (c) Compound 5 was administrated subcutaneously in four different doses: 118.7, 237.5; 475 and 950 mg/kg [18–19]. (d) Finasteride was used as known control and was injected subcutaneously as described in (c). The behavior of each group of animals was observed at 1, 12, 24, 48 and 72 h prior to injections (Fig. 1).
3. Results 3.1. Synthesis of the steroidal derivatives Compounds 5–11 were prepared from the commercially available 16-dehydropregnenolone acetate 4 (Fig. 2). Compound 4 on treatment with m-chloroperbenzoic acid in chloroform afforded the 5,6-epoxy derivative 5. The hydroxycarbonyl derivative 6 was obtained when the epoxy compound 5 was treated with chromium trioxide in acetone–water. Elimination of the hydroxyl group in 6 with thionyl chloride,
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Fig. 2. Reaction sequence.
yielded the conjugated ketone 7; when this compound was hydrolyzed with sodium hydroxide in methanol, it afforded the alcohol 8. This compound was oxidized with chromium trioxide in acetone–water to the diene–trienone derivative 9. Esterification of the free hydroxyl group in 8 with benzoic acid and dicyclohexylcarbodiimide afforded the corresponding benzoate ester 10. The p-fluorobenzoate ester 11 was obtained when the alcohol 8 was esterified in the same manner with p-fluorobenzoic acid.
velopment of inhibition plots. Unmodified [3 H]T was identified from control incubations which did not contain tissue. 3.3. Determination of 50% inhibitory concentration of the novel compounds in hamster prostate The concentrations of compounds 3, 5, 6, 7, 9, 10 and 11 required for inhibiting 5␣-reductase activity by 50% (IC50 ) were determined from the inhibition curves using different concentrations of the steroids and are shown in Table 1.
3.2. In vitro experiments The in vitro biological activity of compounds 3, 5, 6, 7, 9, 10 and 11 was determined in gonadectomized hamster and human prostate glands homogenized and centrifuged to obtain the prostatic enzyme. The activity of hamster and human 5␣-reductases was assessed incubating the enzymatic fractions with 2 nM [3 H]T. The dichloromethane extracts from castrated male hamster and human prostates were subjected to TLC analysis. The zone corresponding to DHT standard (Rf value of 0.67) of the experimental chromatogram was cut off, soaked in Ultima Gold and the radioactivity determined. This result was considered to be 100% of the activity of 5␣-reductase for de-
Table 1 The IC50 values were determined for finasteride and the synthesized steroids 5, 6, 7, 9, 10, 11 with human and gonadectomized hamsters prostatic 5␣reductase; they represent the concentration of the steroid that inhibits 50% of 5␣-reductase activity and were determined as described in Section 2 Treatment
Hamster prostatic enzyme IC50 (M)
Human prostatic enzyme IC50 (M)
T+3 T+5 T+6 T+7 T+9 T + 10 T + 11
1.0 × 10−8 6.5 × 10−11 1.0 × 10−4 6.0 × 10−11 9.2 × 10−10 6.0 × 10−11 2.0 × 10−4
8.5 × 10−9 6.3 × 10−11 2.0 × 10−4 6.5 × 10−11 8.5 × 10−10 7.0 × 10−11 3.5.0 × 10−4
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The IC50 values obtained from hamster and human prostate’s 5␣-reductase enzyme for compounds 5, 7, 9 and 10, were similar; however they were much lower than that for finasteride 3, thus indicating a much higher 5␣-reductase inhibitory activity (Table 1). Since the weight of the prostate gland depends on the 5␣reduced androgens [20], it was important to determine the effect of the new steroidal compounds 5, 6, 7, 9, 10 and 11 on the activity of 5␣-reductase enzyme. 3.4. In vivo experiments After castration, the weight of the male hamster prostates decreased (p < 0.005) compared to that of the normal glands. Treatment with vehicle alone did not change this condition, whereas s.c. injections of 200 g of T for 6 days significantly increased (p < 0.005) the weight of the prostate glands in castrated male hamsters (Table 2). When testosterone (T) and finasteride were injected together, the weight of the prostate decreased (p < 0.005) as compared to testosterone-treated animals (Table 2). However, when the same experiment was carried out with compounds, 5, 6, 7, 9, 10 and 11 the weight of the prostate decreased significantly (P < 0.005) only in the case of steroid 5 thus showing a comparable anti-androgenic effect to that of finasteride 3 (Table 2). The experiment with DHT showed that compound 5 is only an inhibitor of the 5␣-reductase and not an antagonist to the androgen receptor. This fact is demonstrated by the prostate gland weights such as: control 39.1 ± 8.5 mg: DHTstimulated weight 110.7 mg, and compound 5 plus DHT experiment of 101.7 ± 16.5 mg. 3.5. Toxicological assay The lethal dose 50 was determined in Artemia salina model. The results from Experiment 1, indicated that in the control experiment, all of the Artemias remained alive, whereas in Table 2 Weight of prostate glands ±standard deviations from animals receiving different s.c. treatments for 6 days Treatment
Prostate weight (mg)
Control T T+3 T+5 T+6 T+7 T+9 T + 10 T + 11
48.5 87.6 55.0 57.8 78.7 77.1 74.1 76.0 75.0
± ± ± ± ± ± ± ± ±
9.59 10.2 10.7 4.8 14.8 15.6 15.9 18.6 15.0
Significant differences (p < 0.05) between T and control (vehicle) and T plus finasteride or T plus 5, were observed. Three groups of animals were kept as control; one was injected with 200 l of sesame oil (Vehicle), the second one with 200 g of T and the third one T with plus finasteride 3 for 6 days (see Section 2).
Fig. 3. Determination of lethal doses 50 (LC50) by Redd-Muench plot. Cultures of Artemia salina were kept in the presence of the vehicle and compound 5. The two plots (dead and survivors) join in the LC50 point, and represents the doses required for the Artemia salina population.
cultures containing different concentrations of compound 5 the results indicated that the low and high concentrations exhibited the highest number of survivors. These data show very clearly that the epoxy compound does not exhibit any toxicological effects, since most of the crustacean remained alive. An increase of the concentration of compound 5 in the culture medium produced some dead crustacea (Fig. 3). This experiment shows very clearly that only very elevated concentrations of compound 5 resulted in the death of some crustacea. 3.5.1. In vivo acute evaluation of lethal dose 50 in mice No change was observed in the intact as well as in the vehicle controls. Compound 5 as well as finasteride did not produce any lethal and toxicological effects at the doses used. 4. Discussion In this paper we assessed the 5␣-reductase inhibitory activity of six new pregnane derivatives 5, 6, 7, 9, 10 and 11 by comparing their IC50 values. The results from this study with hamster and human prostate enzymes showed very clearly that compounds 5, 7, 9 and 10 (Table 1) are much better inhibitors for the enzyme 5␣-reductase than the presently used Proscar 3 (finasteride). These results confirm also the similarity of both hamster and human 5␣-reductase enzymes. The fact that the optimum pH for hamster’s 5␣-reductase activity at 2 nM is 7 [13] whereas that for human is 6.5 at the same concentration indicates very clearly that some differences in their activity are present. Although the active steroidal derivatives 5, 7, 9 and 10 have different functional groups, they all have one moiety in common, the ␣,-unsaturated C-20-carbonyl group. Accessible electrophilic -carbon of an ␣,-unsaturated carbonyl moiety reacts very readily with a variety of nu-
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cleophiles to form Michael aducts. Several years ago, we postulated that the inhibition of the enzyme 5␣-reductase is based on the Michael type addition reaction of the enzyme 5␣-reductase to the steroidal enone or dienone [12,21]. The results from this study indicated that the first step in the inhibition of the enzyme 5␣-reductase consists in the formation of an enzyme-steroid activated complex. In a subsequent step, the nucleophilic portion of the enzyme (amino group) attacks the conjugated double bond of the steroid in a Michael type addition reaction to form an irreversible adducts. This concept explains very well the high 5␣-reductase inhibitory activity of the steroidal derivatives 5, 7, 9 and 10 which are Michael acceptors (Table 1). In steroid 5, (epoxy compound) the nucleophilic enzyme attacks the electrophilic center at C-6 and this process inhibits the enzyme. Although the chemical structure of the compounds with low activity (6 and 11) is very similar to that of the active steroids 5, 7, 9 and 10, apparently the presence of a fluorine atom in 11 and a hydroxyl group in compound 6 reduces the solubility of these steroidal derivatives and thus diminishes the effectiveness of the Michael type addition reaction. This mechanism has recently been confirmed for the inhibition of the enzyme ubiquitin isopeptidase by prostaglandins containing the cyclopentenone moiety [22]. In vivo experiments demonstrated that only steroid 5 (weight of the prostate gland 57.8 mg) has a comparable activity to the commercially available finasteride 3 (weight of the prostate gland 55.0 mg) (Table 2). All other compounds 6, 7, 9, 10 and 11 showed a weak activity as compared to testosterone treated animals. The in vivo activity of steroid 5 in blocking T-induced prostate weight gain is not due to the antagonist effect on the androgen receptor, since this steroid did not block the DHT-induced prostate weight gain. In order to develop a good inhibitor for the enzyme 5␣reductase which could be used for the treatment of human androgen-dependent disorders such as: benign prostatic hyperplasia and prostate cancer, it is imperative to carry out preclinical evaluation with the synthesized compounds 5, 6, 7, 9, 10 and 11 in vitro and also with animals. The data from our studies indicate that the in vivo efficiency of the 5␣-reductase inhibitors may depend not only on its affinity for the enzyme but also on the rate and extend of absorption, as well as the half life of the parent compound and active metabolites in the prostate. Our studies have also provided evidence about the similarity between hamster and human 5␣-reductase enzyme behavior in the presence of inhibitors at their corresponding pH and concentration optimum. The result of the toxicological assays carried out with Artemia salina cultures showed very clearly that steroid 5 did not produce any toxicological and lethal effects, and as a consequence of this it could be considered a save compound with high pharmacological potential. Since compound 5 is in human prostate enzyme about 100 times more active as 5␣-reductase inhibitor than the commercially available Proscar 3 (finasteride) (Table 1) and does
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not produce any toxicological effects in the near future, this compound will be evaluated on a clinical level. The synthesis of this compound is very simple and much cheaper than that of finasteride 3, and this steroidal derivative could represent a magnificent alternative for the treatment of androgendependent diseases.
Acknowledgements The authors gratefully acknowledge the financial support of CONACYT and DGAPA for projects 33450-M and 200301. The authors would like to thank Alejandro OrtizOsornio and Ruth Bustamante-Garc´ıa for technical assistance in toxicological assays.
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