Steroids an Overview on 5-Reductase Inhibitors

Steroids 75 (2010) 109–153

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Steroids

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Review An overview on 5␣-reductase inhibitors

Saurabh Aggarwal a, Suresh Thareja a, Abhilasha Verma a, Tilak Raj Bhardwaj a,b, Manoj Kumar a,∗ a University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India b I. S. F College of Pharmacy, Ferozepur Road, Moga, Punjab 142001, India article info abstract

Article history: Benign prostatic hyperplasia (BPH) is the noncancerous proliferation of the prostate gland associated Received 13 July 2009 with benign prostatic obstruction and lower urinary tract symptoms (LUTS) such as frequency, hesitancy, Received in revised form 9 October 2009 urgency, etc. Its prevalence increases with age affecting around 70% by the age of 70 years. High activity of Accepted 20 October 2009 5␣-reductase enzyme in humans results in excessive dihydrotestosterone levels in peripheral tissues and Available online 30 October 2009 hence suppression of androgen action by 5␣-reductase inhibitors is a logical treatment for BPH as they inhibit the conversion of testosterone to dihydrotestosterone. Finasteride (13) was the ﬁrst steroidal 5␣- Keywords: reductase inhibitor approved by U.S. Food and Drug Administration (USFDA). In human it decreases the 5␣-Reductase inhibitors Androgens prostatic DHT level by 70–90% and reduces the prostatic size. Dutasteride (27) another related analogue ␣ Azasteroids has been approved in 2002. Unlike Finasteride, Dutasteride is a competitive inhibitor of both 5 -reductase Testosterone type I and type II isozymes, reduced DHT levels >90% following 1 year of oral administration. A number BPH of classes of non-steroidal inhibitors of 5␣-reductase have also been synthesized generally by removing 5␣-Dihydrotestosterone one or more rings from the azasteroidal structure or by an early non-steroidal lead (ONO-3805) (261). In this review all categories of inhibitors of 5␣-reductase have been covered. © 2009 Elsevier Inc. All rights reserved.

Contents

1. Introduction ...... 110 2. Enzyme 5␣-reductase ...... 110 3. Steroidal 5␣-reductase inhibitors ...... 111 4. 2- and 3-Azasteroids ...... 111 5. 4-Azasteroids...... 112 6. 6-Azasteroids...... 121 7. 7-Azasteroids...... 124 8. 8-Azasteroids...... 124 9. 9-Azasteroids...... 124 10. 19-Nor-10-azasteroids ...... 124 11. 11-, 12a-, 13-Azasteroids ...... 125 12. 15- and 16-Azasteroids ...... 125 13. 17- and 17a-Aza-D-homosteroids ...... 125 14. Diazasteroids ...... 125 15. 11,13,15-Triazasteroids...... 126 16. B,D-Dihomo-azasteroids...... 126 17. Des-AB-azasteroids ...... 126 18. Steroidal 3-carboxylic/phosphonic/phosphinic acids ...... 127 19. Diazoketone steroids ...... 128 20. 4-Substituted steroids ...... 128 21. Steroidal oximes ...... 129 22. Steroidal tetrahydrooxazin-2-ones...... 129

∗ Corresponding author. Tel.: +91 172 2534115; fax: +91 172 2534112. E-mail addresses: [email protected] (S. Aggarwal), manoj [email protected] (M. Kumar).

23. 16-Substituted steroids ...... 130 24. 6-Methylene steroidal derivatives ...... 130 25. Seco steroids ...... 131 26. Derivatives of natural substrate: pregnane ...... 131 27. Non-steroidal 5␣-reductase inhibitors ...... 135 28. Mimics of 4-azasteroids: benzo[f]quinolinones...... 135 29. Pyridones, quinolinones and piperidines ...... 136 30. Mimics of 6-azasteroids: benzo[c] quinolinones...... 139 31. Mimics of 10-azasteroids: benzo[c] quinolizinones ...... 139 32. Non-steroidal aryl acids ...... 141 33. Bisubstrate inhibitors ...... 146 34. Miscellaneous non-steroidal inhibitors ...... 147 35. Conclusion and future ahead ...... 149 Acknowledgements ...... 149 References ...... 149

1. Introduction values for relevant compounds have been compared according to the molecular class. The values given are not comparable across Benign prostatic hyperplasia (BPH) is the noncancerous growth the studies and in each comparison a standard taken in the study of the prostate gland resulting due to over-proliferation of the is mentioned. stromal and glandular elements of the prostate [1]. It is caused due to the augmented levels of the androgen dihydrotestosterone 2. Enzyme 5␣-reductase (DHT). In BPH, microscopic foci within specific regions of the prostate grows to form macroscopic nodules which eventually dis- A significant correlation between the androgens and prostate is place the normal prostatic tissue and results into the uretheral well known. Testicular androgens constitute the most important compression. This compression resulting due to increased cell pro- mitogenic factor in vivo for the prostate [11]. Normal circulating liferation and/or impaired apoptosis causes physical enlargement levels of androgens are required for the maintenance of structural of the prostate gland and is referred to as static component. In function, growth and integrity of the prostate tissue. However, addition dynamic component involves sympathetic nerve stim- androgens have no direct effect on prostatic epithelial cells in ulation causing contraction of prostatic and uretheral smooth culture [12]. Androgens enhance the production of many growth muscle which results into outflow obstruction [2]. Despite several factors in the prostate tissue in vivo through a complex cell to hypotheses the molecular trigger for BPH remains unknown [3]. cell interaction involving both epithelial and stromal prostatic cells The incidence of BPH is about 70% at 70 years of age and becomes [13]. Androgen signalling cascade involves the synthesis of T (1)in nearly universal with advancing age. Clinically, BPH causes a con- testes and adrenal glands which gets peripherally converted to DHT stellation of symptoms known as lower urinary tract symptoms (2). DHT formed gets transferred to the target tissues and binds to (LUTS). The hallmarks of the LUTS include frequency, hesitancy, the target receptor with consequent modulation of gene expression urgency, nocturia, slow urinary stream and incomplete emptying [14]. Both T and DHT bind to and activate the androgen receptor [4]. Earlier the choice of treatment in BPH was watchful waiting, (AR), but DHT shows a higher affinity leading to different kinetic transurethral resection of the prostate (TURP) or open prostate- processes. DHT dissociates from AR protein much more slowly than ctomy but due to the invasive nature and potential side effects its precursor. Therefore, at a given time ARs are occupied by DHT many medical therapies have emerged involving the suppression of much more than by testosterone. T is converted to DHT by a steroid androgen stimulation of prostatic growth [5]. These therapies delay 5␣-reductase enzyme (3-oxo-steroid-4-ene dehydrogenase {E.C. or eliminate the requirement of surgery. 5␣-Reductase enzyme 1.3.99.5}) which is a system of two membrane bound nicotinamide has emerged as a target for the pharmaceutical treatment of BPH dinucleotide phosphate (NADPH) dependent enzymes at the level as abnormally high activity of the enzyme in humans results in of prostatic stromal and basal cells. This has led to the develop- excessive DHT levels in peripheral tissues and hence suppression ment of steroidal and non-steroidal 5␣-reductase inhibitors as they of androgen action by 5␣-reductase inhibitors is a logical treatment inhibit the conversion of T (1) to DHT (2) as shown in Fig. 1 [15–17]. for BPH [6]. A large number of molecules have been synthesized as Thus 5␣-reductase dictates the cellular availability of DHT to pro- potential 5␣-reductase inhibitors over the years. Several analogues static epithelial cells and consequently modulates its growth. may also act as androgen receptor antagonists by preventing the The proposed chemical mechanism of T (1) reduction to DHT (2) natural ligands of the androgen receptor such as testosterone (T) (1) by 5␣-reductase catalysis involves the formation of a binary com- and DHT (2) from binding to the receptor. Combination of these two plex between the enzyme and NADPH, followed by the formation of categories of inhibitors may provide effective androgen receptor a ternary complex with the substrate T. A delocalized carbocation is blockage without undesirable side effects of castrate testosterone levels on muscle and bone mass, energy level and libido which are of particular concern [7]. Some earlier reports have been there cover- ing various aspects of 5␣-reductase enzyme and inhibitors [8–10] but a comprehensive review of each category and structural features required for 5␣-reductase inhibitory activity were missing. This review is an attempt to cover all categories of inhibitors of 5␣- reductase with an aim to list the most potent compounds of each category along with the special structural requirements that led to 5␣-reductase inhibitory activity and in vitro data obtained from the evaluation of steroidal and non-steroidal compounds that have ␣ been tested as inhibitors of 5␣-reductase. In particular IC50 and Ki Fig. 1. Site of action of 5 -reductase inhibitors. S. Aggarwal et al. / Steroids 75 (2010) 109–153 111

Fig. 2. 5␣-Reduction of 3-keto-4 steroids.

formed due to the activation of the enone system by a strong inter- More recently with the development of genome-wide gene action with an electrophilic residue (E+) present in the active site. expression profile analyses a third type of 5␣-reductase enzyme Enolate of DHT is formed by the direct hydride transfer from NADPH (type III) has been identified in hormone-refractory prostate can- to the ␣ face of the delocalized carbocation leading to a selective cer cells (HRPC) [26]. This enzyme also converts T to DHT in HRPC reduction at C-5. This enolate which is coordinated with NADP+ on cells in a similar way to type I enzyme and was found to be active the ␣ face, is attacked by a proton on the ␤-face at C-4 giving the at pH 6.9 [27]. Type III isozyme has been recognized as a ubiquitous ternary complex E-NADP+-DHT. Binary NADP+–enzyme complex enzyme in mammals. Northern blot and real time RT-PCR analyses is formed after departure of DHT and finally the release of NADP+ have identified this enzyme in both androgen and non-androgen leaves the free enzyme for further catalytic cycles. Three differ- target human tissues such as pancreas, brain, prostate cancer cell ent types of inhibitors could be conceived according to the kinetic lines, skin and adipose tissues [28]. mechanism of testosterone reduction: type A inhibitors which are Based on their structure 5␣-reductase inhibitors are discussed competitive with the cofactor (NADPH) and the substrate (T) and below as steroidal and non-steroidal inhibitors. interact with the free enzyme; type B inhibitors which are competitive with the substrate and fit the enzyme–NADPH complex, and type C inhibitors which fits the enzyme–NADP+ complex exhibiting 3. Steroidal 5␣-reductase inhibitors uncompetitive mechanism versus the substrate [8,18]. More potent inhibitors of steroid 5␣-reductase have been found As the only information available about the 5␣-reductase among the transition state analogues as molecules mimicking the isozymes is their primary sequence estimated from c-DNAs the transition state of the enzymatic processes exhibit a greater binding design of novel inhibitors is affected. Due to the unstable nature to the enzyme and hence produce greater inhibition. The enzyme of enzyme during purification its crystal structure is not known. 5␣-reductase binds the 3-keto-4 steroids in such a way that the The first inhibitors have been therefore designed by modifying the carbonyl group is brought into vicinity of a positively charged structure of natural substrates, including the substitution of one centre on the enzyme whereby the conjugated ketone becomes carbon atom of the rings of the steroids by a heteroatom such as activated as shown in Fig. 2. nitrogen thereby forming azasteroids. Singh and coworkers [29,30] A hydride ion can then be transferred from the coenzyme as well as other groups have published comprehensive reviews on NADPH to the 5␣-position of the steroid. The resulting enolate is biological activity of azasteroids [31]. Azasteroidal compounds hav- protonated at the axial 4␤-position by the solvent and the product ing nitrogens at various positions have also been covered in this is released. This evidence for protonation was based on the model review. However, their 5␣-reductase inhibitory activity has either studies with the Penicillium decumbens 5␣-reductase enzyme [19]. not been done or they are devoid of activity. Some azasteroids have Modern methods of molecular biology had assisted in identi- been found to be 5␣-reductase inhibitors. In the following section fying two types of 5␣-reductase enzyme: type I and type II from azasteroidal inhibitors have been discussed depending upon the human and rat prostatic complimentary deoxyribonucleic acid position of nitrogen in the steroidal nucleus, i.e. nuclear azasteroids. (cDNA) libraries and the structures of both genes were elucidated at the beginning of this decade [20,21]. The type I enzyme is not the major species expressed in the prostate and is present mainly in the 4. 2- and 3-Azasteroids hair follicles and peripheral skin whereas type II 5␣-reductase is the major isozyme in genital tissues and a deletion in the gene leads Although some of the 3-azasteroids were synthesized by to male pseudohermaphroditism [22,23]. Type I enzyme is consti- Doorenbos and Wu [32] and Mazur [33] in the early 1960s but tutively expressed in the brain and in adulthood appears mainly Anderson and Liao in 1968 reported for the first time that steroidal ␣ localized in the myelin membranes and has a catabolic rather than N-oxido-3-aza-1,3,5(10)-triene is a good inhibitor of enzyme 5 - an activating role in the brain while type II enzyme is transiently reductase [34]. Haffner in 1994, reported the synthesis of some expressed in the prenatal period and in males its expression is novel 3-pyridyl-N-oxide steroids (3 and 4) [35] which mimic the controlled by androgens and appears to be confined in the hypotha- enolate or enol like transition state of the enzyme–substrate com- lamus and in the hippocampus after stress hence type II enzyme plex. might participate in the perinatal differentiation of brain towards a male pattern [24]. The two isozymes differ in the constitution of amino acids as well as molecular weight. The type I isozymes is active at pH 6–9 while type II is active at pH 5.5. The two isozymes also differ in the location of the gene structure while type I is located at 5p15 while type II is located at 2p22 although they had same gene structure [8,25]. The comparison of the properties of two isozymes is summarized in the Table 1: 112 S. Aggarwal et al. / Steroids 75 (2010) 109–153

Table 1 Comparison of properties of 5␣-reductase isozymes.a

Properties Type I 5␣-reductase Type II 5␣-reductase

Size 259 amino acids 245 amino acids Molecular weight 29,462 Da 27,000 Da Optimal pH 6–8.5 5.0–5.5 Biochemical properties Hydrophobic Hydrophobic Gene location SRD5A1, 5p15 SRD5A2, 2p23 Gene properties 5 exons, 4 introns 5 exons, 4 introns

In vitro inhibition by Finasteride Ki ≥ 300 nM Ki = 3–5 nM Localization (in tissues) Sebaceous glands of the skin, sweat glands, dermal papilla cells Prostate, genital skin, epididymis, seminal ﬁbroblasts from all areas, epidermal keratinocytes, follicular vesicles keratinocytes Selectivity to the inhibitors Inhibitors with 4-methyl-4-aza functionality are very potent 4-Aza, 6-aza and charged 3-substituents derivatives are highly selective

a Ref. Li et al. [8].

N-Oxide steroids (3) and (4) were assayed against both type I and possessing these features act as competitive inhibitors of testos- type II 5␣-reductase and proved to be potent inhibitors of type II terone 5␣-reductase, therefore, all of them could be regarded as a 5␣-reductase with the Ki (␮M) being 0.031 and 0.104, respectively. substrate of the enzyme 4-en-3-one steroids [38]. 4-Androsten-3- In 2003, Robinson et al. reported the synthesis of various 2- and one-17-carboxylic acid (11) was identiﬁed as a potent inhibitor of 3-azasteroidal derivatives (5–10) as effective and stable transition 5␣-reductase. state 5␣-reductase inhibitors [36].

All the synthesised 2- and 3-azasteroids (5–10) were evaluated for human 5␣-reductase inhibition. Amines (6 and 10) showed poor inhibitory activity against both type I and type II isozymes whereas lactams (5 and 9) displayed only marginal improvement against type II isozymes. However, nitrones (7 and 8) showed signiﬁcant enhancement in biological activity.

5. 4-Azasteroids

4-Azasteroids is one of the extensively studied and clinically used classes of azasteroidal 5␣-reductase inhibitors. Voigt et al. in 1970, screened a large number of steroids including 23 steroidal hormones for their ability to inhibit the conversion of T (1) into DHT (2) by a crude cell free enzyme system isolated from rat ventral It has been reported to be a competitive inhibitor of the enzyme prostate [37]. In 1973, series of effective 5␣-reductase inhibitors and showed 87.7% inhibition for the microsomal enzyme of human were synthesized and evaluated. From the studies it was estab- skin. None of the compounds from this series could be shown lished that the key structural requirements for the 5␣-reductase to interfere with in vivo conversion of the dihydrotestosterone, inhibitory activity were presence of 4-en-3-one function and 17␤- because of their rapid conversion into the inactive 4,5-dihydro form side chain having one or more oxygen functionalities. Molecules by the enzyme. S. Aggarwal et al. / Steroids 75 (2010) 109–153 113

Fig. 3. Basic SAR of 4-azasteroids.

In search for a nonreducible inhibitor of 5␣-reductase, Merck aza-5␣-androst-1-en-3-one, MK-906, Finasteride (13) was found and Co. in 1980 reported series of 4-azasteroids where C-4 of 3- to be the best and extensively studied. Finasteride (13) was a oxo-5␣-steroids was replaced by nitrogen. The studies showed potent inhibitor of 5␣-reductase type II with only weak in vitro that there was not only an increase in the 5␣-reductase inhibitory activity versus 5␣-reductase type I having IC50 value of 9.4 and activity but also retention of the in vivo activity [39,40]. Therefore, 410 nM, respectively. At clinical dose, 5 mg/day, it caused 65–80% azasteroids were designed to mimic the putative enzyme-bound lowering of plasma DHT levels [50]. Finasteride (13) was the enolate intermediate by incorporating sp2-hybridized center at C- ﬁrst drug to be approved in U.S. for BPH. Long-term studies have 3 and C-4. Thus a lactam was introduced in the ring A of the steroids demonstrated that there is a sustained improvement in BPH to mimic the enol transition state of the enzyme–NADPH–substrate disease and reduction in the prostate speciﬁc antigen (PSA) level [51].

It was reported by Merck as well as Glaxo in 1996 that Finas- (E.NADPH.S) complex. Substitution at C-17 has been found to teride (13) and close analogues are mechanism-based inactivators enhance potency by binding to a lipophilic pocket on the enzyme. of 5␣-reductase II. Although it is accepted as an alternate substrate These competitive inhibitors strongly interact with the enzyme at and is ultimately reduced to dihydrofinasteride (15), this pro- the active site and on other hand unlike the substrate cannot be fur- ceeds through an enzyme-bound NADP-dihydrofinasteride adduct. ther reduced to 5␣-metabolites thus have in vivo inhibitory activity Initially it was believed that Finasteride (13) act as a transition [41]. The steroidal pharmacophore provides an anchor between the state mimic whereby confirmation of the A-ring lactam closely key A-ring lactam and the C-17 substituent while the former acts as mimics the enol form of transition state of 5␣-reduced testos- a transition state mimic of intermediate enolate, the latter signifi- terone but now it is understood that the most likely cause of cantly enhances potency via binding at a pocket largely lipophilic in the slow offset inhibition is rate-limiting hydride transfer from nature. The key 4-aza-3-oxo-5␣-androstane pharmacophore and NADPH to the 1-double bond of Finasteride (13). In the case of the basic structure activity relationship (SAR) is outlined below the 1,2-ene-containing Finasteride (13), reduction of C1 enables (Fig. 3) [42,43]. the nucleophilic attack of C2 on the nicotinamide C4. This aber- Taking into consideration that substitution at C-17␤- could dra- rant reduction results in the formation of lactam enolate which matically affect the potency; a large number of modifications were is not positioned for efficient protonation by the enzyme. Instead carried out to find potent inhibitors [44–46]. the enolate is trapped by the electrophilic pyridinium cation of the 4-MA {17␤-N,N-diethylcarbamoyl-4-methyl-4-aza-5␣- NADP, yielding a covalent adduct to the cofactor and to the pro- androstan-3-one} (12) was found to be a potent dual inhibitor tein (Fig. 4). This dihydrofinasteride–NADP adduct is a remarkably of both human 5␣-reductase isozymes having IC50 value of potent bisubstrate analog inhibitor and it binds to the free enzyme ␣ 1.9 nM against human 5 -reductase II and 1.7 nM against human with a second-order rate constant equal to kcat/Km for turnover of ␣ −13 5 -reductase I, however, it was withdrawn from the clinical T(1) and has a dissociation constant Ki ≤ 1 × 10 M. Finasteride developments due to hepatic toxicity and lack of selectivity over (13) is also a mechanism-based inhibitor of the human skin (type 3␤-hydroxy steroid dehydrogenase enzyme [47–49]. Out of the I) isozyme, but it is processed with a much smaller second-order ␤ × 3 −1 −1 series its unsaturated analogue 17 -(N-tert-butylcarbamoyl)-4- rate constant, ki/Ki =3 10 M s , which attenuates its activ- 114 S. Aggarwal et al. / Steroids 75 (2010) 109–153

Fig. 4. Mechanism of Finasteride inhibition of 5␣-reductase.

Table 2 Table 3 In vitro screening of compounds 20–27 against type I and type II human steroid In vitro screening of compounds 28–33 against human and rat prostatic 5␣- 5␣-reductase. reductase.

Compounds Human 5␣-reductase Human 5␣-reductase Compounds Human 5␣-reductase, Rat 5␣-reductase, IC50 type I, IC50 (nM) type II, IC50 (nM) IC50 (nM) (nM) 20 20 0.2 28 41 83 21 350 24.6 29 212 – 22 >1000 25.2 Turosteride (30)55 53 23 120.2 0.4 31 381 227 24 5.6 <0.1 32 1218 1611 25 14.0 <0.1 33 1553 1154 26 8.1 0.2 4-MA (12)2837 Dutasteride (27) 2.4 0.5 Finasteride (13) 52 <0.1

Bakshi et al. reported a series of 4-aza-3-oxo-5␣-androstene- ity against this isozyme in vivo. Indeed, Merck has demonstrated l7␤-N-aryl-carboxamides as dual inhibitors of human type I and ␣ the presence of (14c) in the inhibited form of 5 -reductase type I type II steroid 5␣-reductases [56]. Some of these compounds [52–54]. were found to be potent inhibitors of both isozymes. Variation of Weintraub et al. in 1985 reported 20-(hydroxymethyl)-4- the C-17 amide substituent on the 4-aza-3-androstane skeleton ␣ methyl-4-aza-2-oxa-5 -pregnan-3-ones and their corresponding has resulted into a fruitful search of the potent dual azasteroid 3-thiones (16–19). These compounds were tested in vitro for inhibitors (20–26). inhibition of testosterone 5␣-reductase and were found to be −7 weak inhibitors with Kis in the 10 range. It was argued that replacement of C-2 in the steroid nucleus by oxygen in the case of 4-aza-3-oxo-steroids would convert it to a urethane (17) from a lactam (16), respectively, thereby enhancing the polarity at C-3 carbonyl, and its afﬁnity for the enzyme active site [55]. S. Aggarwal et al. / Steroids 75 (2010) 109–153 115

Dutasteride (GG745), 17␤-N-{2,5-bis(trifluoromethyl)- phenyl)}-3-oxo-4-aza-5␣-androst-1-ene-17-carboxamide (27) had emerged as the most potent dual inhibitor from this group of placebo with the exception of a modestly elevated incidence (Table 2) [57]. It has been approved by U.S. FDA in 2002, for the of impotence and decreased libido compared with placebo. Also symptomatic treatment of BPH [58,59]. Unlike Finasteride (13), Dutasteride did not clinically significantly impact bone metabolism Dutasteride (27) is a competitive inhibitor of both 5␣-reductase markers, bone mineral density or lipid levels [65]. type I and 5␣-reductase type II isozymes, reduced dihydrotestosterone levels >90% following one year oral administration [60].It is also a time dependent inhibitor as Finasteride (13) and it forms a stable complex with a slow rate of dissociation constant and does not bind to the androgen receptor [61]. By reducing DHT level, it reduces the size of enlarged prostate, so improving the urinary flow rate. It is about 60 times more potent than Finasteride (13) and has been shown to decrease the risk of acute urinary retention and BPH related surgery [62–64]. This greater degree of suppression of serum DHT has been found to correlate with the intraprostatic DHT suppression. Dual inhibition of 5␣-reductase is more beneficial than selective type II inhibition as dual inhibition does not allow the escape of DHT which can formed through type I mediated synthesis thus providing greater efficacy as DHT levels are suppressed to a great extent. Long-term studies have shown that Dutasteride (27) a dual inhibitor is well tolerated during daily In a programme aimed at searching for novel 5␣-reductase use for up to 2 years. It had a tolerability profile comparable to that inhibitors a series of C-17-acylurea-substituted 4-azasteroids (28–33)(Table 3) were synthesized by Di Salle et al. in early 1990s exploiting the tolerance of functionality at this position. Signifi- cantly greater potency was found with the derivative containing C-4 methyl group and a saturated A-ring [66]. 116 S. Aggarwal et al. / Steroids 75 (2010) 109–153

Table 4 Observation that selectivity of inhibitors can be increased In vitro screening of compounds 38–43 against type I and type II human steroid against type I isozyme by making correct choice of hydrophobic 5␣-reductase. substituent at C-17 position led to development of various 17␤-(N- Compounds IC50 (nM) or % inhibition at 100nM (given in parenthesis) ureylene-N,N -disubstituted)-4-methyl-4-aza-3-one 5␣-reductase ␣ Human 5␣-reductase Human 5␣-reductase derivatives (Table 4)(38–43)as5 -reductase inhibitors as they type I (transfected 293 type II (transfected have potent selectivity against 5␣-reductase type I enzyme. Aza- cells) SW-13 cells) steroids with N-cyclopropyl ring exhibit potent inhibitory activity 38 27.3 ± 3.4 (20 ± 3.1) against type I 5␣-reductase. Increase in the chain length from N - 39 5.3 ± 1.1 (46.3 ± 1.3) ethyl to N-butyl the compound showed strong inhibitory activity 40 24.2 ± 1 (4.9 ± 0.2) while branching of alkyl chain decreased potency of compounds 41 1.3 ± 0.64 (56.3 ± 4.5) ± ± and introduction of 1,2-double bond signiﬁcantly reduced the 42 31.5 6.0 (48.7 4.2) 43 11.5 ± 1.9 (39.7 ± 0.4) activity. Replacement of N -alkyl chain with phenyl moiety gave Finasteride (13) 262 ± 43.2 8.5 ± 0.4 the most active compound (41) of the series [68].

Table 5 In vitro screening of compounds 44–49 against type I and type II human 5␣- reductase.

Compounds Type I, IC50 (nM) Type II, IC50 (nM) 44 3.05 ± 0.296 >100 45 0.91 ± 0.236 >100 46 2.19 ± 0.476 >100 47 2.35 ± 0.421 >100 48 9.57 ± 1.745 14 ± 1.11 49 16.9 ± 3.911 18.4 ± 1.541 Finasteride (13) 26.3 ± 4.784 4.53 ± 0.96

Table 6 In vitro screening of compounds 50–54 against type I and type II human 5␣- reductase.

Compounds Type I, IC50 (nM) Type II, IC50 (nM)

50 1.77 ± 0.343 1000 > IC50 > 100 51 2.42 ± 0.409 1000 ␤ 52 2.93 ± 2.158 3.75 ± 1.977 From the studies that 17 -carboxamides at C-17 position 53 10.5 ± 2.739 582 have pronounced activity of 5␣-reductase and possess andro- ± 54 5.44 1.067 1000 > IC50 > 100 gen receptor activities, a number of 17␤-(N-alkyl/aryl formamido) ± ± Finasteride (13) 26.3 4.784 4.53 0.96 (44–49) and 17␤-[(N-alkyl/aryl)alkyl)aryl amido]-3-oxo-4-aza- 5␣-steroids (50–54) were prepared from 17␤-hydroxy-4-aza- One of the most potent compound of this group, Turosteride steroids and were evaluated as 5␣-reductase inhibitors by Li et al. (30), a close analogue of 4-MA (12), but unlike 4-MA was found (Tables 5 and 6). Structure activity relationship indicated that 5␣- to be devoid of binding at the rat androgen receptor and a weak reductase type I enzyme has preference for N-substituted linear inhibitor of 3␤-hydroxy steroid dehydrogenase [67]. alkyl side chain of 4–5 carbon atoms. N-Amyl substituted 17␤- Other azasteroids which retained 5␣-reductase inhibitory formamide (45) was found to be one of the most promising inhibitor activity are 2-substituted (34), A-homo- (35) and 19-nor- (36) of 5␣-reductase type I while N-heptyl (48) and N-octyl (49) showed analogues [41]. dual inhibition of both isozymes of 5␣-reductase (Table 5). S. Aggarwal et al. / Steroids 75 (2010) 109–153 117

Table 7 In vitro screening of compounds 55–61 against type I and type II human 5␣- reductase.

Compounds IC50 (nM)

Type 1 Type 2

55 1.7 218 56 5.7 330 57 1.6 298 58 2.0 125 59 0.6 147 60 8.4 5.4 MK-386 (61) 0.9 154 Finasteride (13) 52 <0.1

Table 8 In vitro screening of compound 62 against human and rat 5␣-reductase inhibition.a

Compounds IC50 (nM)

Human Rats

FCE 27837 (62) 51(3) 60(3) ␤ Similarly in series of 17 -[(N-alkyl/aryl) alkyl) arylamido] Finasteride 51(6) 32(5) derivatives (Table 6) exhibited highly potent inhibitory activity for a Number of assays in parentheses. human 5␣-reductase type I [69].

Various 7␤-substituted derivatives have also been prepared. Preliminary screening of the compounds as inhibitors of 5␣- reductase from human scalp and prostate revealed that the (Table 7) [71]. presence of 7␤-methyl substitution in ring B, presence of cholesterol type side chain at C-17 and ketone functionalities at C-3 in 4-azasteroids resulted in potent selective inhibitor against 5␣- reductase type I [70].

During 1995, some 17␤-hydroxy-17␣-(␻-hydroxy/haloalkyn- l-yl)-4-methyl-4-aza-3-oxo-5␣-androst-1-ene-3-ones were synthesised by Li et al. and their antiandrogenic activity was reported [72]. Salle et al. have reported synthesis and 5␣- reductase inhibitory properties of various 4-azasteroids with ﬂuoro substituted 17␤ amidic side chains [73] and further investigated FCE 27837 (N-[1,1,1-triﬂuoro-2-oxobut-3-yl]-3-oxo-4-aza- 5␣-androst-1-ene-17␤-carboxamide) (62), for its endocrinological 4,7␤-Dimethyl-4-aza-5␣-cholestan-3-one (MK 386) (61) properties in comparison with those of Finasteride (13)(Table 8) emerged as one of the most potent inhibitor of type I 5␣-reductase [74]. 118 S. Aggarwal et al. / Steroids 75 (2010) 109–153

Table 9 Table 10 In vitro screening of compounds 63–66 against human steroid 5␣-reductase. In vitro screening of compounds 67–69 against human steroid 5␣-reductase.

Compounds Human 5␣-reductase inhibition Ki (nM) Compounds Human 5␣-reductase inhibition Ki (nM) 63 2.6 67 0.5 64 1.8 68 2.3 65 2.2 69 3.2 66 5.1 Table 11 In vitro screening of compounds 70–72 against human steroid 5␣-reductase.

Compounds Human 5␣-reductase, IC50 (nM) 70 16 71 8 72 14

In order to have speciﬁc and dual inhibitors of 5␣-reductase Labrie and associates synthesized several steroids having lactam in ring A and substitution at 17␤ position. Several of the compounds were found active (Tables 9 and 10) [75].

Panzeri et al. reported the syntheses of several 17␤- substituted 4-aza-5␣-androstan-3-one carboxamides with unsaturation between C-1 and C-2 (70–72). These were found to be highly potent against human 5␣-reductase enzyme (Table 11) [76]. S. Aggarwal et al. / Steroids 75 (2010) 109–153 119

Table 12 Table 14 In vitro screening of compound 73 against human steroid 5␣-reductase. In vitro screening of compounds 75–79 against human steroid 5␣-reductase.

Compounds Type I, IC50 (nM) Type II, IC50 (nM) Compounds Rat 5␣-reductase Human 5␣-reductase relative %inhibition at inhibitory potency to MK-906 73 36 ± 9 3.3 ± 1.2 10−8 M (MK-906 = 1) Finasteride (13) 470 ± 41 8.5 ± 1.2 75 50 0.55 76 74 1.6 77 74 2.9 78 39 <0.1 Table 13 79 33 1.0 ␣ In vitro screening of compound 74 against rat and human steroid 5 -reductase. Finasteride (13) 28 1.0

Compounds IC50 (nM)

Rat prostate 5␣-reductase Human prostate 5␣-reductase

74 36 262 Table 15 Finasteride (13)11 18 In vitro screening of compounds 80–85 against human steroid 5␣-reductase.

Compounds IC50 (nM)

Type I Type II

80 13 0.2 In 1996, Giudici et al. reported the synthesis of FCE 28260 81 420 20 (73) [(22R,S)-N-(1,1,1-triﬂuoro-2-phenylprop-2-yl)-3-oxo-4-aza- 82 30 210 ␣ ␤ 83 120 50 5 -androst-1-ene-17 -carboxamide] (Table 12) as a potent dual 84 410 15 inhibitor of both 5␣-reductase isozymes and it was found to cause 85 511 74% reduction in the DHT levels [77]. Finasteride (13) 52 <0.1

Table 16 In vitro screening of compounds 86–89 against human steroid 5␣-reductase.

Compounds IC50 (nM)

Type I Type II

86 30 210 87 610 88 27 89 620 Finasteride (13) 52 <0.1

CIBA-GEIGY Ltd. reported the synthesis of CGP53153 (N-(2-cyano-2-propyl)-3-oxo-4-aza-5␣-androst-1-ene-17␤- carboxamide) (74)(Table 13), a novel inhibitor of 5␣-reductase and structurally related to Finasteride (13) was found to be 10 times more potent than Finasteride in reducing prostate weight of rat [78].

In 1996, Ishibashi et al. reported the synthesis of various 11␣-acetoxy, 11␣-hydroxy, 11␤-hydroxy and 11-oxo substituted 4-aza-5␣-androstane analogues (75–79) with a diphenylmethyl- Merck in 1997 reported the synthesis of several 4-aza 5␣- carbamoyl moiety at C-17 and their evaluation. Compounds with an androstan-3-one 17␤-(N-substituted carboxamides) (80–85)as 11␤-hydroxy or 11-oxo showed inhibitory activities comparable to potent human type II 5␣-reductase inhibitors. From the studies it Finasteride (13). The 4-methyl 11␤-hydroxy-4-aza-5␣-androstane was indicated that the 17-amide N substituent included aromatic derivative (77) was found to be most potent against rat and human residue potent dual inhibitors of type I and type II 5␣-reductase enzyme and more active than Finasteride (13)(Table 14) [79]. were obtained (Table 15). 120 S. Aggarwal et al. / Steroids 75 (2010) 109–153

The addition of N4-methyl substituent in A-ring increases human androgen receptor afﬁnity while addition of unsaturation to the A-ring (1) increased human androgen receptor binding. The unsubstituted carbanilides in the 1-N4-methyl series showed some selectivity for type I 5␣-reductase over type II enzyme. Whereas addition of aryl substitution at the 2-position increased type II 5␣-reductase binding, thus providing dual inhibitors with excellent human androgen receptor binding. Compound (87)was found to be the most potent inhibitor from this series (Table 16) [80].

Fig. 5. SAR of 6-azasteroids.

In 2000, Lerner and coworkers reported the synthesis of haptens 91(a,b) and 92(a,b) which belongs to 4-aza steroids. The resulting 5␣-dihydrotestosterone was shown to be the more potent intracellular hormone [82].

In 1998, Salle et al. reported synthesis of a novel compound PNU 157706 [N-(1,1,1,3,3,3-hexaﬂuorophenylpropyl)-3-oxo-4-aza-5␣- Table 17 ␣ a androst-1-ene-17␤-carboxamide] (90) as a potent dual type I and In vitro screening of compound 90 against human steroid 5 -reductase. type II 5␣-reductase inhibitor. PNU 157706 (90) was found to Compounds Type I Type II reduce prostate weight 16-fold than Finasteride (13) while the ED50 90 3.9 ± 0.1 1.8 ± 0.3 values being 0.12 and 1.9 mg/kg/day, respectively (Table 17) [81]. Finasteride (13) 313 ± 74 11.3 ± 2.6

a Results are the mean ± SE of 3 separate assays. Incubations were performed in the presence of 3 or 1 ␮M[3H] testosterone, for type I or type II isozyme, respectively.

Table 18 In vitro screening of compounds 94–101 against human steroid 5␣-reductase.

Compounds Type I 5␣-reductase, Type II

IC50 (nM) 5␣-reductase, IC50 (nM)

94 750 1.5 95 180 2.3 96 51 9 97 97 2.1 98 40 3.9 99 1,300 5.7 100 14,000 1.8 101 3,500 3.4 Finasteride (13) 150 0.18 S. Aggarwal et al. / Steroids 75 (2010) 109–153 121

Menzenbach et al. reported the synthesis of several 17- methylene-4-azasteroids as inhibitors of 5␣-reductase. Azaestra- none II (93) was found to be inhibitor of 5␣-reductase with tural and charge polarization features of the transition state for the × −10 × −10 IC50 =34 10 (for prostate) and IC50 =25 10 (for seminal enzyme catalyzed transfer of hydride from NADPH to testosterone. vesicle) [83]. The higher reduction potential of ketoenamine compared to that of ␣,␤-unsaturated ketone prevents these compounds from acting as substrates for 5␣-reductase and they show slow offset inhibition instead of irreversible as shown by 4-azasteroids [53]. Structure activity relationship has also been reported by Frye and associates at C-4, N-6 and C-17 carbamoyl (Fig. 5) [43,84]. Initially a set of N-6, C-1, C-2, C-4 substituted derivatives of 6- aza-androst-4-en-3-ones (94–101) were prepared to explore the structure activity relationship of A- and B-rings versus type I and type II 5␣-reductase (Table 18).

6. 6-Azasteroids

Glaxo was the ﬁrst to report design of 6-azasteroidal inhibitors based on the 3-keto-4-en-6-amine functionality to mimic the struc-

Table 19 In vitro screening of compounds 102–114 against human steroid 5␣-reductase.

Compounds Type I 5␣-reductase, Type II 5␣-reductase,

IC50 (nM) IC50 (nM) 102 12 1.4 103 9 <0.10 104 4.5 <0.10 105 30 <0.10 106 150 3.2 107 3.6 <0.10 Methylation at N-6 (95) and substitution of C-4 with small 108 6.9 <0.10 lipophilic groups such as Cl (96), Br (97) and CH3 (98) increases type 109 20 0.16 I5␣-reductase activity selectivity 4-fold while type I 5␣-reductase 110 20 0.12 activity was decreased by unsaturation (99), 1,2 cycloproponation 111 12 <0.10 112 20 0.40 (100) and C-2-methylation (101). 113 1.0 <0.10 By careful optimization of the C-17 substituent along with com- 114 4.0 <0.10 bining A- and B-ring substitutions, potent dual inhibitors of both Finasteride (13) 150 0.18 isozymes of 5␣ reductase were obtained (102–114)(Table 19) [84]. 122 S. Aggarwal et al. / Steroids 75 (2010) 109–153

It was found that optimizing C-17 group resulted in 5␣- reductase type I inhibitory activity in 103 with 5–7-fold increase of activity in 105. Swapping a methyl ester (106) for an admantyl A variety of C-17 amide-substituted 6-aza-androst-4-en-3-ones ester (108) provides selectivity towards 5␣-reductase I. Preparation were prepared and evaluated against human type I and type II of analogues of 103 resulted in compounds (109–112) which were steroid 5␣-reductase in order to optimize potency versus both found to be 16–200-fold selectivity towards type I 5␣-reductase. isozymes of 5␣-reductase. Out of the study two series of potent and Compounds (103, 111, 113 and 114) having ketone at C-17 proved selective C-17 amides were discovered, 2,5-disubstituted anilides extremely potent inhibitors of type I 5␣-reductase. Out of the and (arylcycloalky1) amides (115–121)(Table 20). Evaluation of group, compound (105) demonstrated efficacy equivalent to Finas- some optimal compounds from this series in a chronic castrated teride (13) in a castrated rat model of DHT dependent prostate rat model of 5␣-reductase inhibitor induced prostate involution, growth. In general, large lipophilic groups at C-17 provides selec- and pharmacokinetic measurements identified compounds (117, tivity against 5␣-reductase I. 118, 120 and 121) with good in vivo efficacy and half-life in the dog [85]. S. Aggarwal et al. / Steroids 75 (2010) 109–153 123

B-Homologated analogue of 17␤-N, N-diethylcarboxy-6-aza- Table 20 ␣ androst-4-en-3-one (122) has also been found to be potent In vitro screening of compounds 115–121 against human steroid 5 -reductase. inhibitor of 5␣-reductase inhibitor with IC50 = 318 nM [86]. Compounds Type I 5␣-reductase, Type II 5␣-reductase, IC50 (nM) IC50 (nM) 115 4.2 <0.1 116 4.6 <0.1 117 8.8 <0.1 118 1.3 <0.1 119 4.0 <0.1 120 6.8 <0.1 121 0.6 <0.1 Finasteride (13) 150 0.18

Fang and Sharp in 1996 synthesized several 6-azaandrostenones of the general structure (127)as5␣-reductase inhibitors [89].

Bergmann et al. synthesised 7-substituted ␦-4-6-azasteroid derivatives (123)as5␣-reductase inhibitors [87]. But activity data was not reported for these compounds.

16␤-Aryloxy, -alkoxy and heteroaryloxy 6-azasteroids of the general formula as given below were synthesised by Aster et al. and the compounds were found to be potent inhibitors of 5␣-reductase [90]. Compound (128) was found to be potent inhibitor of human 5␣-reductase type I with IC50 in the range of 0.1–1000 nM.

R1 = H or CH3;R2 =CH3;R3 = hydrogen, Alk-R4, X-Alk, C1-6-X- Alk, XCO-Alk, Co-Ar, CO-NH-Ar, CO-NH-Het, etc., where Alk is C1–12 straight or branched alkyl, Ar is phenyl, X is O, N or S, Het is piperidinyl, piperizinyl, pirrolidinyl, pyrrolyl, etc. 6-Azacholesten-3-ones (Table 21) were assayed against both type I and type II 5␣-reductase by Haffner [88]. All three compounds were found to be potent dual inhibitors of 5␣-reductase. Unlike the 4-azasteroids the cholesterol side chain imparts very little selectivity between type I and type II 5␣-reductase. It was also found that C-7 methyl group might provide a potent 5␣- Rahier and Taton have reported synthesis of several novel 6- ␣ reductase I selective compound. The ␣-C-7 methyl diastereomer aza-B-homosteroids but they were not tested for 5 -reductase (124) proved to be 7-fold more active 5␣-reductase I inhibitor inhibitory activity [91]. Later in 2000 and 2001, Wenge et al. than ␤-diastereomer (125). [92] and Xie et al. described the synthesis of 6-azasteroids as 124 S. Aggarwal et al. / Steroids 75 (2010) 109–153

Table 21 Table 22 In vitro screening of compounds 124–126 against human steroid 5␣-reductase. In vitro screening of compounds 129–135 against human steroid 5␣-reductase type II. Compounds Type I 5␣-reductase ki (nM) Type II 5␣-reductase ki (nM) Compounds IC (nM) Finasteride IC (nM) 124 0.8 7.9 50 50rel IC (nM) (IC rel=IC 125 1 1.2 50 50 50 compound/IC 126 1 2.3 50 Finasteride)

129 4600 ± 2990 3.4 ± 2.2 1389 ± 1295 ± ± ± potent phosphatidylinositol phosphalipase C (PI-PLC) inhibitors 129:130 = 5:1 4600 960 4.1 0.7 1123 318 131:132 = 22:1 2900 ± 1190 3 ± 1.4 981 ± 609 [93]. Kasal et al. in 2005 reported an efﬁcient synthesis of 6- 133:134 = 9:1 37 ± 6.7 2.2 ± 0.4 16.9 ± 0.4 aza-allopregnanolone as neurosteroid analogues but not evaluated 133:134 = 3.5:1 150 ± 33 4.3 ± 1.5 34 ± 12 them for 5␣-reductase inhibitory activity [94]. 135 460 ± 229 5.5 ± 2.1 83 ± 52

7. 7-Azasteroids Table 23 In vitro screening of compounds 129–135 against human steroid 5␣-reductase type Some 7-azasteroids were synthesized in early 1970s I in DU-145 cells. [95]. Morzycki and Sicinski reported the synthesis of 6,7- Compounds IC50 (nM) IC50rel (nM) Selectivity 5␣-reductase II: diazacholestane derivatives but they were not evaluated for 5␣-reductase I the 5␣-reductase inhibitory activity [96]. 129:130 = 5:1 263 ± 63 6.7 ± 2 1:17 131:132 = 9:1 127 ± 12 2.8 ± 0.4 1:1 8. 8-Azasteroids 135 1134 ± 288 24.4 ± 7 2.5:1

Several 8-azasteroids have been synthesized [97] and discussed as antifungal agents [98,99] but none has been reported as 5␣- reductase inhibitor.

9. 9-Azasteroids

No work has been published on 9-aza steroids as 5␣-reductase inhibitor although some fungicides have been known from this category [100].

10. 19-Nor-10-azasteroids The inhibitory potency of these compounds depends on the presence of bridgehead N-10 atom conjugated with 4-en-3-one On the basis of the molecular model of active site for type II moiety in A-ring, unsaturation in C-ring and substituent at C- isozyme and to increase the activity and selectivity of compounds 17 position. 19-nor-10-aza steroids have low transitional barrier towards both 5␣-reductase type I and 5␣-reductase type II. energy values and more ﬂexible as compared to 6-aza or 4- Guarna et al. synthesized a novel class of compounds 19-nor-10- azasteroids [102]. azasteroids (Tables 22 and 23) [101].

Best results were obtained with 9:1 mixture of 9(11) (133) and 8(9) (134)17␤-(N-tert-butyl carbamoyl)-19-nor-10-aza- 4-androsten-3-one as it was found to be a good inhibitor of Some 10a-azasteroids were also synthesized from fusidic acid 5␣-reductase type I and 5␣-reductase type II. The enamine struc- but were not evaluated for 5␣-reductase inhibitors [103]. Guarna ture of ring A of 10-aza-steroid (136) is analogous to that of the et al. also synthesized 17␤-[N-(phenyl) methyl/phenyl-amido] substrate like transition state. The presence of N atom at position substituted 10-azasteroids. Unexpectedly, 5␤-H compounds were 10 increases the nucleophilic character of the carbonyl group and found more active than their 5␣-H counterparts, with (138) stabilizes the carbocation intermediate (137) by delocalization at (IC50 = 279 and 2000 nM toward isoenzymes I and II, respectively) the positive charge. and (139) (IC50 = 913 and 247 nM toward isoenzymes I and II, S. Aggarwal et al. / Steroids 75 (2010) 109–153 125

respectively) being the most potent compounds of the series [104].

11. 11-, 12a-, 13-Azasteroids

Though many 11-azasteroids [105–107], 12a-azasteroids [108] and 13-azasteroids [109] have been prepared but 5␣-reductase Table 24 In vitro screening of compounds 149–152 against inhibitory activities have not been reported. human steroid 5␣-reductase.

␮ 12. 15- and 16-Azasteroids Compounds IC50 ( M) 149 4 Many 15-azasterols have been synthesized as antifungal agents 150 15 151 12 [110–112] but none of them have been evaluated for 5␣-reductase 152 40 inhibitory activity. 16-Azasteroids have been synthesized but none of the compound has evolved as 5␣-reductase inhibitor [113,114]. inhibitors has shown that steroids without side chains can bind to 13. 17- and 17a-Aza-D-homosteroids enzymes with the A-ring of the substance simulating the D-ring of the substrate, while the D-ring emulates the A-ring. This could lead Regan and Hayes, in their exemplary work, have synthe- to 17-D-homo-azasteroids exhibiting same mechanism of action as sized several 17- and 17a-aza-D-homosteroids from several 4-azasteroids. However, up to now, research has been concentrated 17-ketosteroid oximes [115]. But 17a-azasteroids attracted more on 4-azasteroids as 5␣-reductase inhibitors and 17a-azasteroids attention when chandonium diiodide was established as a potent remained unexplored avenue as far as their 5␣-reductase inhibitory neuromuscular blocker [116]. 17 and 17a-Azasteroids have been activity is concerned. Some 17a-azasteroids (149–152) evaluated found to possess numerous biological activities like gamma amino for 5␣-reductase inhibitory activity are summarized in the Table 24 butyric acid (GABA) receptor antagonistic [117–119], antifungal [124]. [120], antineoplastic, mutagenic [121,122] and anti-inﬂammatory

14. Diazasteroids activity [123]. The most interesting aspect concerning 17-D-homo- azasteroids is the possibility of “inverted action” or “backbinding” Eberbach and coworkers reported in 1996 a novel access to ␣ as proposed by MacDonald et al. [124]. Their proposition was based 4,13-diazasteroid derivatives but they were not evaluated for 5 - on the fact that the steroids have the potential to bind in two reductase activity [128]. In the same year Stuart et al. first reported ␣ orientations in the active site of various metabolizing enzymes. 4,17-diazasteroids as potential inhibitors of 5 -reductase. The Marcus and Talalay [125] first reported that 3(17) ␤-hydroxysteroid Finasteride 17-aza-isomer (153) proved to be potent inhibitor ␣ dehydrogenase converts both T (1) and dehydroepiandrosterone of 5 -reductase II although less active than Finasteride (13) and (141) to androst-4-ene-3,17-dione (140)(Fig. 6a). Other examples its congeners. 4-Methylation (154) lowered the inhibition of the ␣ 1(2) of enzyme inhibition by inverted steroids have also appeared. The 5 -reductase II enzyme. Removal of unsaturation led to the ␣ oxiranes 143 and 144 were found to be active site-directed, irre- formation of compound (155) that is dual inhibitor of 5 -reductase versible inhibitors of 3-oxo-5-steroid isomerase (Fig. 6b) [126] type I and type II and 4-methylation of 155 led to the increase in 5(6) and the bromoacetates 147 and 148 act as affinity labels for estra- activity in 156. While compound with (157) showed only a ␣ diol 17␤-dehydrogenase (Fig. 6c) [127]. Research on 5␣-reductase moderate inhibition of 5 -reductase II activity (Table 25) [129]. 126 S. Aggarwal et al. / Steroids 75 (2010) 109–153

8,13-Diaza steroids were also synthesized by Göndös et al. in 1998 but not evaluated for 5␣-reductase inhibitory activity [130].

15. 11,13,15-Triazasteroids

Hirota et al. reported in 1995 the synthesis of 11,13,15- triazasteroid derivatives to investigate antidepressive activity. These analogues were also evaluated for anti-platelet aggregation activity and some derivatives exhibited positive action but no 5␣-reductase activity has been investigated in these categories of steroids [131].

16. B,D-Dihomo-azasteroids

Several steroidal B,D-dihomolactam have been synthesized and evaluated for antitumour activity but no 5␣-reductase activity has been reported from this group till date [132,133].

17. Des-AB-azasteroids

Trehan et al. synthesized des-AB-azasteroids but 5␣-reductase activity studies were not done [134]. Steroidal 5␣-reductase inhibitors which are extranuclear, i.e. in which nitrogen is not the part of steroidal nucleus but forms part of the side chain or attached group have also been explored as 5␣- reductase inhibitors, therefore are discussed next.

Table 25 In vitro screening of compounds 153–157 against human steroid 5␣-reductase.

Compounds Type I 5␣-reductase, Type II 5␣-reductase,

IC50 (nM) IC50 (nM) 153 765(±70) 10.3(±1.1) 154 477(±29) 174(±42) 155 2200(±140) 40.1(±2.8) 156 28.0(±2.1) 3.6(±0.3) 157 ∼7000 52.0(±7.8) 4-MAa (12) 6.4(±0.2) 0.4(±0.04) Fig. 6. (a) Action of 3(17) ␤-hydroxysteroid dehydrogenase, (b) action of 3-oxo-5- a N,N-Diethyl-3-oxo-4-methyl-4-aza-5␣-androstrane-17␤-carboxamide (4-MA) steroid isomerase, and (c) action of estradiol 17␤-dehydrogenase. was used as a standard reference. S. Aggarwal et al. / Steroids 75 (2010) 109–153 127

Table 26 unsaturation in the B- and D-rings, and in the C-17 carboxamide In vitro screening of compounds 158–166 alkyl groups. Despite lacking C-19 methyl group these compounds against human steroid 5␣-reductase. were found to be potent inhibitors of 5␣-reductase [138].

Compounds Ki,app (nM) 158 30 159 7–18 160 26 161 7–12 162 7 163 30–36 164 32 165 35 166 50

18. Steroidal 3-carboxylic/phosphonic/phosphinic acids

A number of 3-androstene-3-carboxylic acids (158–166) (Table 26) were designed to mimic the putative enzyme-bound enolate intermediate by incorporating sp2-hybridized centers at C-3 and C-4 and, most critically, an anionic carboxylic acid at Nitro derivatives also showed interesting structure activity C-3 as a charged replacement for the enolate oxyanion. Because relationship patterns compared to carboxylic acids, compound of presumably favorable electrostatic interaction between the (172) was found to be potent competitive inhibitor by binding to carboxylate and the positively charged oxidized cofactor, the E-NADPH complex [139]. The sulphonic acid (173) [140], phospho- acrylate preferentially binds in a ternary complex with enzyme nic acid (174) and phosphinic acid (175) also proved to be the ␣ ␣ and NADP+, which leads to the uncompetitive kinetic mechanism potent inhibitors of human 5 -reductase but not so of rat 5 - [135–137]. Activity is enhanced in analogues possessing an addi- reductase [141]. The afﬁnities of phosphonic acid are relatively tional unsaturation at C-5 (162–167) along with 3-unsaturation. less than phosphinic acid derivatives because the increased bulk At C-17, diisopropyl (158) and pivalyl (163) amides were optimal. at the 3-substituent, leading to a steric intolerance for binding to Epristeride (SK&F 105657) (163) entered the clinical trials for enzyme. Overall 3-phosphosteroids were weaker inhibitors than treatment of BPH and is a potent inhibitor of 5␣-reductase II while their corresponding steroidal-3-carboxy steroids. The function of a weak inhibitor of 5␣-reductase I.

A series of estratriene-3-carboxylic acids containing an aromatic the C-3-moiety is presumably to act as an H-bond acceptor from a A-ring had also been synthesized (167–171)(Table 27) with struc- residue in the enzyme, which would normally donate a hydrogen tural variations in the C-2 and C-4 substituents, in the degrees of bond to stabilize the enolate. Since the negatively charged groups − (CO2 ) or isosteres of the carboxylate (NO2) best mimic this interaction it indicates that a pKa-matched H-bond with a Lys or Table 27 In vitro screening of compounds 167–171 Arg donor may be operative. In addition, an interaction between against human steroid 5␣-reductase. the negatively charged C-3-moiety and the positively charged NADP+ cofactor after the enzyme has turned over substrate is Compounds K (nM) i,app possible, especially if the cofactor lies directly under the A-ring of 167 20 the steroidal skeleton in the transition state and mimics thereof. 168 30 Compound 176 was not as active due to the presence of alcoholic 169 10 170 35 group as it was not able to provide sufﬁcient negative charge and 171 36 hence was a weak inhibitor of rat and human 5␣-reductase. 128 S. Aggarwal et al. / Steroids 75 (2010) 109–153

19. Diazoketone steroids

The primary evidence of a dramatic increase in the afﬁnity of 5␣- reductase and an inhibitor with a 5-juncture of A-/B-ring and sp2 Table 28 In vitro screening of compounds 180–183 against human 5␣-reductase. hybridization at the C-3 and C-4 positions was obtained from the inhibition with a mechanism-based inhibitor (5,20R)-4-diazo-21- Compounds Human 5␣-reductase Human 5␣ reductase hydroxy-20-methyl-pregn-6-en-3-one (177) (RMI-18,341). Dia- type I (transfected 293 type II (transfected cells) (IC nM) SW-13 cells) (IC nM) zoketone (177) had been reported to be a potent time-dependent 50 50 180 2.9 inhibitor with a Ki of 35 nM (time-dependency is considered indica- 181 709 437 tive of irreversibility) [142]. 182 >1000 192 183 981 387 Finasteride (13) 218 8.47

Finasteride (13). Out of these 4-cyano compounds were found to be potent inhibitors of 5␣-reductase type II enzyme and substitution with groups like thiol led to decreased activity. This series of compounds were also found to be potent androgen antagonists [144]. Fei et al. carried out the synthesis of some novel 4-triﬂuoromethylsteroids and proposed them as novel 5␣-reductase inhibitors. Out of the series 4-triﬂuoromethyl-N-(t- butyl)-4-androsten-17␤-carboxamide (184) emerged as the most A mechanism of inhibition was proposed that the protonation potent inhibitor being 4 times more active than Finasteride (13) steps implicated in the normal enzymatic transformation activates the diazoketone functionality to a diazonium ion that could further alkylate some nucleophilic residue at the active site [143].

20. 4-Substituted steroids [145]. 4-Cyanoprogesterone (185) was also found to be a potent inhibitor of both rat and human 5␣-reductase enzymes (IC50 val- The observation that an excellent inhibitor possessed a con- ues = 0.045 and 0.050 ␮M, respectively). The mechanism of action of jugated system (sp2–sp2–sp2) at C-3, C-4, and C-5 positions 4-cyano steroidal inhibitor was assumed to be the transition state of A-ring of steroids together with a lipophilic group at C-17, inhibitor because on reduction by the enzyme compound would a range of 4-substituted-3-oxo-4-androstene-17(-carboxamides form a stable 5–3-enol that would remain tightly bound to the (180–183)(Table 28) were prepared and compared with the active site [146,147]. S. Aggarwal et al. / Steroids 75 (2010) 109–153 129

21. Steroidal oximes Transferring the oxime group from positions 20 to 21 caused an increased selectivity toward type II isozyme. Z-21- A number of pregnenolone (186–190) and progesterone Hydroxyiminopregn-4-en-3-one (195) was found to be a potential (191–194)-based steroids were synthesized bearing a oxime group inhibitor of the type II (IC50 = 1.95 ␮M against human type I and connected directly or via a spacer to the steroidal D-ring, capable to 0.30 ␮M against human type II). form a coordinate bond with haeme iron of enzyme 5␣-reductase. In contrast to the pregnenolone derivatives which showed no inhibition of 5␣-reductase isozyme I and II, progesterone derivatives possessed marked inhibition towards type II.

Inhibitory potency of synthesized compounds against target enzyme using whole cell assay revealed that C-20 oxime (191) displayed strong inhibition against both isozymes (IC50 = 1.63 against human type I and 0.58 ␮M against human type II). Unsaturation in ring D (192 and 193) in conjugation with oxime group further enhances inhibitory activity. Almost complete loss of activity has been found toward type I in the derivative with keto group at C-6 (194).

However, none of the compounds showed activity near to that of the reference drug Finasteride (13) (IC50 values being 45 and 3 nM for type I and type II, respectively, in the corresponding study) [148].

22. Steroidal tetrahydrooxazin-2-ones

Wölfing et al. synthesized a novel series of steroidal tetrahydrooxazin-2-ones (196–201) containing heterocycles 130 S. Aggarwal et al. / Steroids 75 (2010) 109–153 involving O and N heteroatoms at position 17␤ of androst-4-en- Certain 19-nor analogues have also been synthesized in order to 3-one, respectively, as 5␣-reductase inhibitors. The IC50 values of improve the inhibitory activity in 16-methylated derivatives. TSAA- compounds vary between 270 and 600 nM. The relative inhibitory 291 (16-ethyl-17␤-hydroxy-4-estren-3-one) (208) was found to be effect of the unsubsituted N-phenyl compound 196 is 0.20. Con- the first anti-androgen known to have dual action of competitive cerning the effects of substituents at position 4 of the phenyl inhibition of 5␣-reductase activity and androgen receptor complex ring in 196, the introduction of an ethyl (197) or ethoxy (199) formation. It showed a Ki of 1400 nM to the purified nuclei from rat group resulted in a weak enhancement of 5␣-reductase inhibition. prostatic tissues [151]. Substitution with halogens (200 and 201) or methoxy (198) caused lowering of inhibition ability [149].

24. 6-Methylene steroidal derivatives

2,3␣-Tetrahydrofuran-2-spiro-17-(6-methylene-4- androsten-3-one) (209; L612,710) is a potent time-dependent inhibitor which causes the highest percentage of inhibition (81%) of rat prostatic 5␣-reductase enzyme. The structure activity relationship showed that 3-oxo-4-ene functionally was essential Steroid 5␣-reductase inhibitors that do not contain any nitrogen to the inhibitory activity and that substituents at C-17 influenced either as part of ring or extranuclear but yet found to inhibit 5␣- the inhibitory potency. The presence of the C-19 methyl group was reductase are discussed next. not essential to the activity. The A-ring appeared to interact with the entire active site of the enzyme. Furthermore, the affinity of an 23. 16-Substituted steroids inhibitor to the enzyme was greatly enhanced by the introduction of a methylene group at C-6 whereas activity was completely lost A series of 16-methyl substituted derivatives of androst-4- with a large radical such as iodomethylene group at C-7. Thus a ene and estr-4-ene originally prepared as antiandrogens, were series of 6-methylene steroids were prepared and examined as tested for their inhibitory activity on rat and human prostatic irreversible inhibitors of rat prostatic 5␣-reductase [152]. Another 5␣-reductase. The inhibitory activity data indicated that IC50 6-methylene steroid that is potent inhibitor due to priming increases in sequence in derivatives bearing 16␣-methyl (203), of its dienone group by electrophilic activation toward nucle- 16␤-methyl (204) and 16,16-dimethyl substituents (205). Acy- ophilic attack at the 6-methylene group is having structure (210) lation of 17-hydroxy group significantly increases the inhibitory [153]. potency (IC50 (207) = 4.8 nM, IC50 (206) = 23.5 nM in rat prostate and IC50 (207) = 0.52 nM, IC50 (206) = 0.62 nM in human prostate). Overall, in human prostate homogenates IC50 varies between 0.6 and 120 ␮m while in rat prostate it ranges from 1.6 to 1000 ␮M. This shows enzyme of human prostate is more sensitive than that of rat prostate to methyl substituted compounds. Overall 16-methyl steroids were found to be weak inhibitors both in rat and human enzymes compared to the existing ones [150]. S. Aggarwal et al. / Steroids 75 (2010) 109–153 131

25. Seco steroids

(4R)-5,10-Seco-estra-4,5-diene-3,10,17-trione (211) and (4R)- 5,10-seco-19-nor-pregna-4,5-diene-3,10,20-trione (212) were 26. Derivatives of natural substrate: pregnane ﬁrst found to be non-competitive and possibly irreversible ␣ inhibitors of epididymal 5 -reductase. Radiographic crystallog- As a consequence of the important observation that pro- raphy studies of both compounds showed that the conjugated gesterone and deoxycortisone inhibits the synthesis of dihy- allenic 3-oxo-5,10-secosteriod (211) has a conformation similar to drotestosterone by competing with 4-en-3-one function of the that of the normal tetracyclic steroid dione. Both compounds were testosterone for the 5␣-reductase enzyme it led Voigt and cowork- ␣ non-competitive inhibitors of 5 -reductase and have an afﬁnity ers to synthesize number of progesterone derivatives [37,38]. The label for the enzyme with Ki of 5470 and 980 nM, respectively satisfactory result of 4-cyano-progesterone (185) [146], which [154,155].

possessed marked inhibitory activity for 5␣-reductase enzyme, stimulated great deal of interest to synthesize various 4- and 6-halo-progesterone analogs (213–217). These compounds were found to be potent antiandrogenic in nature when tested against gonadectomized hamster seminal vesicles and were also found to be inhibitors of 5␣-reductase [156–158]. 132 S. Aggarwal et al. / Steroids 75 (2010) 109–153

Bratoeff et al. evaluated the antiandrogenic and 5␣-reductase inhibitory activity of various 16-phenyl substituted-D-homo compounds (218–219), 16-methyl substituted steroids (220–222), 4-bromo compound (223) without a methyl group at C-16 and the epoxy compounds (224–225). Compounds 219, 222 and 223 were found to possess both antiandrogenic and 5␣-reductase inhibitory activity better than the Finasteride (13) [158,159]. The trienones having a more coplanar structure reacts faster with the nucleophilic portion of the enzyme in a Michael type addition reaction to form an irreversible adduct with a concomitant inhibition of the enzyme 5␣-reductase than the dienones.

A range of 4-bromo-17-substituted-4-pregnene-3,20 diones were also synthesized and evaluated as 5␣-reductase inhibitors on gonadectomized hamster seminal vesicle and flank organs. Several new pregnane derivatives were also synthesized and Small diameter of the pigmented flank organ and great reduction evaluated by the conversion of [3H]Tto[3H] DHT in Penicillium in the weight of seminal vesicle has been found with the com- crustosum broths and the conversion of [1,2-14C] sodium acetate pounds having p-fluorobenzoyloxy (226) and p-chlorobenzoyloxy into lipids. Compounds 228 and 229 were found out to be potent (227) indicating that the presence of more electronegative sub- 5␣-reductase inhibitors as they inhibit conversion of T to DHT and stituent at C-17 position (p-halosubstituted phenyl) and halogen at also decreased the incorporation of radiolabeled sodium acetate C-4 enhances the antiandrogenic activity as well as 5␣-reductase into lipids of the flank organs [162]. inhibitory activity [160,161]. S. Aggarwal et al. / Steroids 75 (2010) 109–153 133

receptor. The appropriate homologues extended by a methylene group at C-6 (233–236) showed better activity due to the presence of exocyclic double bond that can react faster with the enzyme in a Michael type addition reaction than the corresponding endo- cyclic diene. Compounds 233 and 234 showed IC50 values of 19 and 100 nM, respectively [164,165].

Cabeza et al. also reported the 5␣-reductase inhibitory activity and the antiandrogenic effect of novel 16-bromo substituted trienedione, 16␤ methyl substituted dienedione and the trienedione (230–232). Compounds 230 and 231 were found to Exhibit 5␣-reductase inhibitory activity higher than the commercially available Finasteride (13) [163].

In compound 237 which is an epoxy compound the nucleophilic enzyme attacked the electrophilic center at C-6 and in this process inhibits the enzyme. Several 3-substituted 4-pregnene-6,20-dione derivatives (238–246) have been synthesized and were evaluated for antiandrogenic as well as 5␣-reductase inhibitory activity. The synthesized steroids have an ␣,␤-unsaturated carbonyl moiety in common. It was found that accessible electrophilic ␤-carbon of an ␣,␤-unsaturated carbonyl moiety reacted very readily with a variety of nucleophiles to form Michael adducts. The ﬁrst step involved the formation of an enzyme-steroid activated complex The in vitro inhibitory activity of some novel progesterone and in a subsequent step the nucleophilic portion of the enzyme derivatives was also determined and they were evaluated as (amino group) attacked the conjugated double bond of the steroid 5␣-reductase inhibitors as well as antagonists for the androgen in a Michael type addition reaction to form an irreversible adducts [166]. 134 S. Aggarwal et al. / Steroids 75 (2010) 109–153

Table 29 The IC50 values of the compounds were found to increase pro- ␣ gressively as the substituent on the phenyl group of the ester In vitro screening of compounds 262–265 against human 5 -reductase. side chain at C-3 became more electropositive, i.e. compound Compounds Type I 5␣-reductase, Type II 5␣-reductase, IC (nM) IC (nM) 241 has an IC50 value of 3.0 nM as compared to compound 50 50 243 which has an IC50 value of 4.0 nM. On the other hand the 262 6500 – compounds (244–246) having saturated 4-pregnene-6,20-dione 263 460 – 264 30 – skeleton for, e.g. 245 exhibited higher IC50 value (3.7 nM) as com- 265 60 – pared to the unsaturated ones (240–243) for, e.g. 241 with a value of 3.0 nM. It was also found that the presence of halogen substituents in ester moiety at C-3 as well as double bond enriched by the presence of halogens in these steroidal derivatives at C-16 increased the binding afﬁnity for the androgen receptor leading to the increase in the inhibition of 5␣-reductase enzyme [167]. as determined by the IC values. Compound 252 was found to be Several new progesterone derivatives were also synthesized by 50 most potent with an IC value of 1.8 nM while compounds 251 the same group having dienone moiety as reported earlier. Some of 50 and 253 showed values 14 and 10 nM, respectively, in comparison them (247–248) were found to have high 5␣-reductase inhibitory to Finasteride (13) having value 8.5 nM. Compound 254 was not activity like 248 showing an IC50 value of 0.5 nM as compared to Finasteride (13) showing an IC50 value of 8.5 nM in the same study [168,169].

Bratoeff et al. synthesized two novel steroidal carbamates (249–250) which are the esters of carbamic acid with substituents at the amino and esters ends (NHRCOOR1) and proposed them that active when compared to halogenated compounds thus as novel class of inhibitors for human and hamster steroid 5␣- further demonstrating the need of a halogen atom in the ester side reductase. These compounds have a longer half-life and are chain [171]. hydrolyzed slowly in the liver due to which they have a better pharmacological activity when compared to the conventional esters. Compound 249 has got a similar IC50 value (10 nM) as that of Finas- teride (8.5 nM) (13) while compound 250 has a higher IC50 value of about 50 nM apparently due to the presence of large bromine atom [170].

Recently, Cabeza et al. synthesized several C-6 substituted and unsubstituted pregnane derivatives as potential 5␣-reductase inhibitors. It has been found that steroids that lack a chlorine atom in C-6 (255–257) exhibited a higher capacity for inhibition of ␣ Working on similar lines some more novel 4,16-pregnadiene- the activity of 5 -reductase (IC50 in the range of 25–63 nM) than 6,20-dione derivatives were synthesized and evaluated as 5␣- the compounds (258–260) having this atom (IC50 in the range reductase inhibitors. In this work, it has been demonstrated that of 920–990 nM in comparison to Finasteride (13) having an IC50 compounds containing chlorine (251), bromine (252), iodine (253) 8.5 nM). The presence of bromine atom in C-6 of compound 261 atoms, and (254); without any substituent in the ester moiety) at C- however doesn’t affect the inhibitory activity of enzyme (IC50 ␣ 3 produce a signiﬁcant decrease of the prostate weight in castrated being 33 nM). Also, the presence of an ester moiety in C-17 animals treated with testosterone (1). Therefore, it was proposed on the steroidal skeleton tends to increase the inhibition of the that the ester moiety at C-3 is functioning as a pharmacophore, activity of the enzyme [172]. S. Aggarwal et al. / Steroids 75 (2010) 109–153 135

␣ 27. Non-steroidal 5 -reductase inhibitors general octahydro derivatives are more potent inhibitors than the corresponding 4a–10a unsaturated compounds (Tables 29 and 30) ␣ A number of classes of non-steroidal inhibitors of 5 -reductase and in both series the potency toward type I 5␣-reductase increases have now been identiﬁed. It was anticipated that the use of non- if an halogen atom is present at position 8 (in particular a Cl atom) steroidal template can decrease the potential interaction with other and a methyl group at position 4; in fact the most potent inhibitor enzyme or receptor of the steroidal endocrine system and can limit of the series is LY191704 (268) with IC50 =8nM(Table 30). This the complexity of target compound synthesis [173]. They have in molecule has progressed into human clinical trials. fact emerged either from the design of compounds mimic of azasteroidal inhibitors, generally by the formal removing of one or more rings from the azasteroidal structure or by early non-steroidal lead (ONO-3805) (261) which was prepared as leukotriene synthesis inhibitor [174] or by high throughput screening. These compounds are generally thought to act all as competitive inhibitors vs. testosterone with exception of the epristeride analogues which are uncompetitive inhibitors. Non-steroidal inhibitors include benzo[f]quinolinones, pyridones and quinolinones which were mimics of 4-azasteroid inhibitors. Benzo[c]quinolinones were synthesized as mimics of 6-azasteroids while benzo[c]quinolizinones were designed as mimics of 10-azasteroids. The most potent and selective inhibitors of human type I 5␣-reductase are found among these classes of compounds. Almost all the other non-steroidal inhibitors can be grouped as carboxylic acid (generally butanoic acid) derivatives which are thought to act as non-competitive inhibitors versus testosterone in analogy to ONO 3805 (261).

28. Mimics of 4-azasteroids: benzo[f]quinolinones The quantitative structure activity relationship study of these Benzo[f]quinolinones were the ﬁrst non-steroidal inhibitors compounds has focused on the effect of 8 substituent on the aro- prepared by the Lilly’s researchers. They were derived by the matic ring, which can be accounted for by its lipophilic character removal of the D-ring from 4-azasteroids and replacing the C-ring [176]. They found that the optimum activity may reside in the prop- with an aromatic one [175]. Most of these compounds are type I erty space around the chlorine substituent. The substitution of the selective, although dual inhibitors can be obtained if an appropri- 8-Cl atom byaF(270) or a Br atom (271) decreased very slightly the ate substitution is present at the position 8 on the aromatic ring. potency. Finally, several kinds of substituents, including complex Two main classes of benzo[f]quinolinones have been described, aromatic groups, were introduced at the position 8 [177], and some the hexahydro derivatives (262–265), which have an unsaturation potent type I selective inhibitors such as compounds 272–274 were at positions 4a–10a, and the octahydro derivatives (266–271). In prepared. 136 S. Aggarwal et al. / Steroids 75 (2010) 109–153

29. Pyridones, quinolinones and piperidines than the corresponding lactams. The aryl acid 277 showed good dual isozyme inhibitory properties with signiﬁcantly enhanced Abell et al. synthesized a number of tricyclic thiolactams type II activity. Bicyclic lactams, in general, were found to be less (275–276), aryl acid (277), bicyclic lactams (278–281) and bicyclic active against type I 5␣-reductase than the tricycles. For example, thiolactam (282) and evaluated them in vitro as inhibitors of type compounds (278–279), lacking the B and D steroidal rings, were I and type II steroid 5␣-reductase (Table 31). Removal of two poor type I 5␣-reductase inhibitors, with the highest potency or more rings from 4-azasteroids resulted in a strong decrease associated to the presence of the Cl atom on the aromatic ring of of potency. The tricyclic thiolactams were found to be selective 279 [178]. A styryl (or azo) substituent dramatically enhances type type I 5␣-reductase inhibitors and in general were less active II activity (and indeed type I activity with the bicycles) (280–281) and provided dual inhibitors of type I and type II. S. Aggarwal et al. / Steroids 75 (2010) 109–153 137

Their poor potency however illustrates the need for both A- and B-rings to be present with the correct fusion pattern for good recog- Table 30 nition at the enzyme active site. A series of 5-phenyl substituted In vitro screening of compounds 266–274 against human 5␣-reductase. 1-methyl-2-pyridones have also been prepared and tested against ␣ Compounds Type I 5␣-reductase, Type II 5␣-reductase, human and rat 5 -reductase type I and type II. Compound 285 ␣ IC50 (nM) IC50 (nM) bearing bulky carboxamide substituents exhibited excellent 5 - ␮ 266 60 – reductase type II inhibitory activity with IC50 value of 10 M [182]. 267 17 – 268 8 10,000 269 11 – 270 35 – 271 35 – 272 59 >10 273 6 1,400 274 6 1,340

Hartmann and coworkers synthesized pyridones of the general formula 283 and 284 where the B- and C-rings of the steroid system have been replaced by an acyclic linker but these compounds display relatively weak activity versus both the rat and human 5␣- reductase isozymes (Ki >20␮M) [179–181]. McCarthy and coworkers have recently synthesized a series of 4-substituted 5-aryl pyridones along with corresponding 1-aryl- pyridone derivatives and tested them against 5␣-reductase type I and type II expressed in transfected human embryonic kidney cells to examine structure activity relationship for the 4 position in pyridones. 138 S. Aggarwal et al. / Steroids 75 (2010) 109–153

Weak inhibition was observed against the type I isozyme for Table 31 ␣ 4-N-substituted acetamide compounds (286–289) while potent In vitro screening of compounds 275–282 against human 5 -reductase. inhibition of type I isozyme was observed for compound 290 having Compounds Type I 5␣-reductase, Type II 5␣-reductase, 4 -benzoyl substituent and also for compounds (291–292) having IC50 (nM) IC50 (nM) or long carbon chain tethers attached to the 4-acetamide (Table 32). percentage inhibition Thus further proving that large hydrophobic groups are tolerated 275 377 13.2% @ 40 ␮M in a region of the active site not involved in the enzymatic reaction 276 183 21.6% @ 40 ␮M 277 152 340 [183]. 278 2477 13.5% @ 40 ␮M Few quinolinone derivatives such as 6-substituted 1H-quinolin- 279 1690 12,350 2-ones (293–294) and 2-methoxy quinolines (295–296) have also 280 302 579 been synthesized. The most active inhibitor for the human type 281 107 617 ␮ II isozyme was 6-[4-(N,N-diisopropylcarbamoyl) phenyl]-1H- 282 3360 14% @ 40 M quinolin-2-one 293 having Ki 800 ± 85 nM, showing mostly competitive inhibitory patterns. A type I selective inhibitor could be identiﬁed with 6-[4-(N,N-diisopropylcarbamoyl) phenyl]- N-methyl-quinolin-2-one 294 (IC50 = 510 nM) but 2-methoxy quinolines were not found to be active [184].

Hartmann and coworkers synthesized and evaluated a series of 2-substituted 4-(4-carboxy- or 4-carboxymethylbenzylidene)- N-acylpiperidines as active steroid 5␣-reductase type II inhibitors. They synthesized several compounds from N-acyl-4-benzylidene- piperidine-4-carboxylic acids. In the dicyclohexylacetyl series, ﬂuorination in the 2-position of the benzene nucleus (297), exchange of the carboxy group by a carboxymethyl moiety (298) and combination of both structural modiﬁcations (299) led to highly active inhibitors of the human 5␣-reductase type II isozyme with IC50 values of 297, 298 and 299 being 11, 6 and 7 nM, respectively, in comparison to Finasteride (13) having value of 5 nM [185].

Earlier Hartmann et al. have reported a series of N-substituted piperidine-4-(benzylidine-4-carboxylic acids) (300–303) as potent non-steroidal dual inhibitors of 5␣-reductase. S. Aggarwal et al. / Steroids 75 (2010) 109–153 139

Table 32 Benzoquinoline derivatives (267–268), novel phenanthridin-3-one ␣ In vitro screening of compounds 286–292 against human 5 -reductase. derivatives (305–307) were synthesized having vinylogous amide Compounds Type I 5␣-reductase Type II 5␣-reductase pharmacophore. (% inhibition at 10 ␮M) (% inhibition at 10 ␮M)

286 8– 287 6– 288 6– 289 3– 290 61 – 291 43 – 292 33 –

Finasteride (13) 453 (IC50 (nM)) 25 (IC50 (nM))

In rat, compounds 300 (IC50 = 3.44 and 0.37 ␮M for type I and type II, respectively) and 302 (IC50 = 0.54 and 0.69 ␮M for type I and type II, respectively) displayed the best inhibition toward both isozymes. Compound 301 showed a strong inhibition toward type II human and rat enzyme (IC50 = 60 and 80 nM) but only a moderate activity versus type I enzyme (IC50 approximately 10 ␮M for rat and human enzyme). Compound 303 (IC in human type II enzyme 50 Although compounds were found to be 5␣-reductase type I being 0.26 ␮M and in rat type II enzyme being 0.29 ␮M) was found selective and poor inhibitors of 5␣-reductase type II but overall to be a moderate dual inhibitor probably due to higher ﬂexibility these compounds did not showed promising inhibitory activity. of the open ring substituent [186]. The potency of compounds was found to increased from 305 Methyl esters of N-(dicyclohexyl)acetyl-piperidine-4- (K 10 ␮M) to 306 (K = 1.1 ␮M) and 307 (K = 0.92 ␮M) this was (benzylidene-4-carboxylic acid) (304) were designed and i i i due to the presence of methyl group on the A-ring corresponding monitored for dual inhibition toward type II isozyme in BPH to the 4-Me of Eli Lilly inhibitors. This effect is due to the presence cell free preparation and for type I isozyme in DU 145 cells. of hydrophobic pocket in the active site of enzyme which is able to Methyl esters, applied as hydrolytically stable precursor drugs to accommodate a small alkyl group located at the position 4 of the facilitate cell permeation, will yield the corresponding carboxylic A-ring of these tricyclic inhibitors [188]. acids as type II inhibitors after hydrolysis in the target organ. The esters themselves stable in human plasma and Caco-2 cells act as potent drug toward 5␣-reductase type I. Thus, dual inhibition ␣ of 5 -reductase type I and type II can be achieved by applying a 31. Mimics of 10-azasteroids: benzo[c] quinolizinones single parent compound [187]. Guarna et al. had synthesized two series of benzo[c]quinolizin- 3-ones as novel inhibitors of human 5␣-reductase type I: 4aH-series with a double bond between the positions 1 and 2 (308–311) and 1H-series with a double bond between the positions 4 and 4a (312–317). The efﬁcacy and selectivity of these compounds have been demonstrated on recombinant human 5␣-reductase type I expressed in CHO cells but they displayed very poor or no inhibition towards 5␣-reductase type II (Table 33). Increased activity of the compounds of 1H-series than those of corresponding inhibitors of 4aH-series has been attributed to the presence of double bond enabling conjugation between carbonyl and nitrogen atom [189–191]. 30. Mimics of 6-azasteroids: benzo[c] quinolinones

On the basis of 6-aza-androst-4-en-3-one derivatives (Fig. 5)in which a vinylogous amide was inserted into a steroid nucleus as a transition state mimic for conversion of T (1) to DHT (2) and Lilly’s

Table 33 In vitro screening of compounds 308–317 against recombinant 5␣-reductase I expressed in CHO cells.

Compounds IC50 (nM) 308 5130 ± 130 309 176 ± 17 310 459 ± 118 311 137 ± 58 312 49 ± 19 313 20 ± 8 314 7.6 ± 0.9 315 14.3 ± 5.9 316 8.5 ± 2.1 ± 317 204 49 Fig. 7. SAR for 4-benzo[c] quinolizin-3-ones. 140 S. Aggarwal et al. / Steroids 75 (2010) 109–153

The presence of a methyl group at position 4 (311, 313, 314, and 316) associated with a substituent at position 8, gave potent compounds in comparison with Finasteride (13) and the known consistent with the observation that the introduction of the same 5␣-reductase I selective inhibitor LY191704 (268). All compounds group on the corresponding position 7 in 4-azasteroids increased inhibited the enzyme through a reversible competitive mechanism. their 5␣-reductase I selectivity [70]. Presence of methyl group at The structure activity relationship carried out on this class espe- position 5 found to decrease the potency of the compounds while cially methyl group at positions 1, 4, 5, 6 and 8 can be summarized introduction of methyl at position 1 as in compound 317 caused as follows (Fig. 7) [191]. only a slight decrease in activity when compared to unsubstituted It was found that presence of substituent at position 8, either compound 312. a chlorine or methyl group, generally increased the potency of In 2001, Guarna et al. studied the effect of C-ring modiﬁcations the inhibitors. Thus compound 309 and 310 were potent were in benzo[c]quinolizin-3-ones. They synthesized several octahydro- more active than unsubstituted compound 308 in 4aH-series. Sim- and decahydrobenzo[c]quinolizin-3-one derivatives containing ilarly in 1H-series compounds 312 and 314 were more potent than partially or fully saturated C-ring. These compounds were found unsubstituted compounds due to presence of 8-chlorine group. The to be selective 5␣-reductase I inhibitors. Benzo[c]quinolizin-3-one introduction of a methyl group at position 4 was found to increase inhibitor lacking the aromatic C-ring but with a double bond at the potency in both series with effect being higher in 1H-series. A 6a–10a 318 displayed an inhibitory potency 345-fold higher than very strong increase of potency is observed in 8-chloro-4-methyl that of the corresponding 6a–10a saturated, trans-fused compound 319 (Table 34) [192].

A 3D-QSAR model correlating the potency of the inhibitors with derivative 314 when compared to 4-methyl unsubstituted com- their physicochemical features using density functional theory pound 312. The substitution with a methyl group at position 6 also (DFT) was also developed for a series of benzo[c]quinolizin-3-ones affects potency in both series with effect being more prominent in derivatives by adding two “non standard” variables (dipole moment 1H-series with compound 315 being more potent than unsubsti- and log P) to the classical electrostatic and steric comparative tuted 312. This effect of methyl substitution at position 6 seems molecular ﬁeld analysis (CoMFA) ﬁelds [193]. With the aim to discover new dual non-steroidal inhibitors of Table 34 5␣-reductase I and II a series of benzo[c]quinolizin-3-ones deriva- In vitro screening of compounds 318 and 319 against recombinant 5␣-reductase I tives (320–325) bearing diverse substituents at position 8 were and II expressed in CHO cells. synthesized in 2005 [194]. They were tested towards 5␣-reductase ␣ ␣ Compounds Type I 5 -reductase, Type II 5 -reductase, IC50 (nM) I and II expressed by Chinese Hamster Ovary cells (CHO 1827 and IC50 (nM) CHO 1829), respectively. It was found out that most potent dual 318a 58 ± 2.1 No inhibition inhibitors were obtained when F atom was introduced on the phe- ± 319 20,000 400 No inhibtion nol moiety of these esters. All compounds displayed inhibition a Mixture of 6a(10a)/10(10a) isomers in 10:1 ratio. towards 5␣-reductase I in the range of 93–165 nM (Table 35). Com- S. Aggarwal et al. / Steroids 75 (2010) 109–153 141 pound 322 was found to be the most potent dual inhibitor of the Table 35 In vitro screening of compounds 320–325 against recombinant 5␣-reductase I and series with IC50 values about 100 nM for both enzymes. II expressed in CHO cells.

Compounds Type I 5␣-reductase (% Type II 5␣-reductase (% inhibition at 10 ␮M) inhibition at 10 ␮M)

320 102 553 321 129 584 322 93 119 323 160 134 324 138 166 325 42 368

Table 36 In vitro screening of compounds 326–330 against recombinant human 5␣-reductase I and II.

Compounds Type I 5␣-reductase (Ki,app) Type II 5␣-reductase (nM) (Ki,app) (nM) 326 315 >10,000 32. Non-steroidal aryl acids 327 320 ∼2,500 328 26 10,000 329 1200 260 Some novel 9,10-dihydrophenanthrene-2-carboxylic acids 330 1900 1,600 (326–328) were prepared by formally removing D-ring from parent androstene carboxylic acid inhibitors and contrary to them, were found to be selective 5␣-reductase I inhibitors. Introduction of a bromine atom at position 7 in compound 328 gave the most Epristeride 163) the selectivity toward 5␣-reductase I was found potent compound of the series. Substitution by chlorine at position to be lost in favor of an increased potency towards 5␣-reductase II 7 in compound 327 does not result in increase in potency as (Table 36) [196]. compared to unsubstituted compound 326. These compounds are supposed to interact with the positively charged enzyme–NADP+ complex in an uncompetitive manner versus testosterone [195].

On removal of two or more rings from the parent steroidal compounds several aryl acids mimics of steroidal carboxylic acids have been synthesized (331–336). Hartmann et al. synthesized several Moreover when double bond was introduced in the B-ring as N-substituted 4-(5-indolyl) benzoic acids but potent and selec- ␣ in compound 329 (formally obtained by removing the D-ring from tive human 5 -reductase I inhibitors were not found from the series. Only compound 331 with IC50 value of 67 nM against human 5␣-reductase I was the most potent inhibitor [197]. Similarly, compound 332 was found to possess IC50 value of 680 nM [198]. 142 S. Aggarwal et al. / Steroids 75 (2010) 109–153

Fig. 9. SAR of indole series. Fig. 8. SAR of benzophenone series. Screening of aryl carboxylate versus the human 5␣-reductase Igarashi et al. synthesized a new series of indole derivatives isozymes by SmithKline-Beecham company led to the discovery as potent human prostatic 5␣-reductase inhibitors. Compounds of two series of selective and potent 5␣-reductase II non-steroidal (333–336) were found to be most potent among the series with inhibitors based on benzophenone and indolecarboxylic acids 4-[(1-benzyl-1H-indol-5-yl) oxy]-3-chlorobenzoic acid 334 having skeleton. In benzophenone series of inhibitors the linker between an IC50 value of 0.44 nM while 3-chloro-4-{1-(4-phenoxybenzoyl)- the A- and B-rings proved to be crucial whereas linker between 1H-indol-5-yl]oxy}benzoic acid 335 showed inhibitory activities B- and C-rings was more tolerant of variation. Both the A-ring for both human and rat prostatic 5␣-reductase with IC50 values of carboxylic acid and C-ring are critical for activity. Compounds 337 2.1 and 73 nM, respectively [199]. and 338 were found to be most potent among the series with Ki,app being 10 and 5 nM, respectively. In indole series there was S. Aggarwal et al. / Steroids 75 (2010) 109–153 143 a strong preference for substitution at the 5- or 6-position of the that (Z)-4-{2-[[3-[1-(4,4-diﬂuorobenzhydryl)indol-5-yl]-2- indole ring. Compounds 339 and 340 were most active among the pentenoyl]-amino]phenoxy}butyric acid (341,KF20405) was the series with Ki,app being 10 and 40 nM, respectively The structure most potent compound having activity about 20 times greater activity relationships of both the series are summarized as below than Finasteride (13) and an IC50 value of 0.48 ± 0.086 nM [201]. (Figs. 8 and 9) [43,200].

Takami et al. synthesized various indole derivatives with varied substituents on the ␣,␤-unsaturated double bond and evaluated them for activity to inhibit rat prostatic 5␣-reductase. Among the various derivatives they found

A novel series of indole and benzimidazole derivatives were also synthesized and evaluated for their inhibitory activity of rat prostatic 5␣-reductase. Among the series, 4-{2-[1-(4,4- dipropylbenzhydryl)indole-5-carboxamido]phenoxy}butyric acid (342) and its benzimidazole analogue (343) showed potent inhibitory activities with IC50 values of 9.6 ± 1.0 and 13 ± 1.5 nM, respectively [202]. 144 S. Aggarwal et al. / Steroids 75 (2010) 109–153

Baston et al. synthesized several 3,4-dihydro-naphthalene-2- carboxylic acids and evaluated them for 5␣-reductase inhibitory Sawada et al. synthesized a novel series of indolizinebutyric activity. The most active inhibitors were 6-[3-(N,N-dicyclohexyl acids with various benzoyl substituents.FK687 (344)(S)-4-[1-[4- aminocarbonyl) phenyl]-3,4-dihydro-naphthalene-2-carboxylic [[1-(4-isobutylphenyl) butyl] oxy] benzoyl] indolizin -3-yl] butyric acid (348) (IC = 0.75 ␮M, human type II; IC = 0.81 ␮M, human acid displayed strongest in vitro inhibitory activity (IC = 4.6 nM) 50 50 50 type I) and 6-[4-(N,N-diisopropylamino-carbonyl) phenyl] against the human enzyme and in vivo inhibitory activity naphthalene-2-carboxylic acid (349) (IC = 0.2 ␮M, human (IC = 1.7 nM) against the castrated young rat model among the 50 50 type II). The latter compound was shown to deactivate the enzyme series [203]. in an uncompetitive manner (Ki =90nM; Km, T = 0.8–1.0 ␮M) similar to the steroidal inhibitor Epristeride (163) [205].

Novel substituted benzoyl benzoic acids and phenylacetic acids were synthesized by Salem et al. based on the template structure 338 and were evaluated for the inhibition of rat and human steroid 5␣-reductase isozymes I and II. The phenylacetic acid derivatives were more potent than the analogous benzoic acids. Bromination in the 4-position of the phenoxy moiety led to the strongest inhibitor of the series against human 5␣-reductase II (352;IC50 =5nM), which was equipotent to Finasteride (13) while compounds 350 In 2002, various N-substituted 4 -biphenyl-4-carboxylic acids (IC50 =23nM) and 351 (IC50 = 27 nM) were also found to potent were synthesized by increasing the conformational ﬂexibility using inhibitors against 5␣-reductase type II [206]. an ether linker between the steroidal A–C-ring mimetics and were tested against human and rat 5␣-reductase type I and type II. Two compounds were found to be most potent with compound 345 showing an IC50 value of 60 nM while 346 showed an IC50 value improved by a factor of 5 from 1.9 to 0.38 ␮M in comparison with the parent biphenyl compound 347 [204]. S. Aggarwal et al. / Steroids 75 (2010) 109–153 145

Compound 358, having 2-hexyloctylamino group, was found to be most potent inhibitor among the compounds with IC50 being 0.60 nM against human and 5.8 nM against rat 5␣-reductase [207]. A novel series of indole-3-alkanoic acids with varied N-benzyl substituents were also synthesized. Amongst these 4-[1-(6,6- dimethyl-6H-dibenzo [b,d] pyran-3-yl) methyl indol-3-yl]-butyric acid (359; FR119680) displayed very high inhibitory activity against rat prostatic 5␣-reductase (IC50 = 5.0 nM) [208].

Kato and coworkers synthesized a series of pyrrole butyric acid derivatives and evaluated them for inhibitory activity on human and rat steroid 5␣-reductase. In case of para-aminobenzoyl pyrrole derivatives (353–356), the introduction of ethyl (354) or isopropyl (355) at C-4 increased the activity [IC50 being 32 and 9.2, respectively], whereas the replacement with carboxyl (356) resulted in compounds with decreased inhibitory activity against human 5␣-reductase enzyme, indicating steric restriction in the binding site of the enzyme. Compound with m-amino benzyl pyrrole (357) moiety was found to be more active than the corresponding para isomer (355) [IC50 = 3.2 nM]. 146 S. Aggarwal et al. / Steroids 75 (2010) 109–153

33. Bisubstrate inhibitors

Ishibashi et al. synthesized a series of novel benzofuran deriva- Igarashi et al. found a novel series of phenoxybenzoic acids tives with both carboxy and 5- or 6-diphenylmethylcarbamoyl ␣ derivatives as potent inhibitors of human prostatic 5 -reductase. groups and their inhibitory activities against rat and human testos- It was found that introduction of a chloro (360), ﬂuoro (361)or terone 5␣-reductase were tested in vitro. The derivatives were more methoxy (362) group at 3-position of benzene ring (R1) leads to for- active against human type I enzyme than against type II enzyme. mation of compounds with high inhibitory activity with IC50 being The 6-carbamoyl derivative such as 365 tended to be more potent 0.87, 0.67 and 0.56 nM, respectively [209]. than the 5-carbamoyl ones such as 366 with 365 being the most potent compound having IC50 value of 37.9, 50 and 340 nM against rat, human type I and human type II isozymes [211].

Later, they also synthesized a series of 2-phenylbenzofuran derivatives with a carbamoyl, alkylamino, or alkyloxy group at the 5 or 6 position of the benzofuran ring. It was found that carbamoyl derivatives had more potent inhibitory activities than the alkylamino or alkyloxy derivatives against the rat enzyme and the 6-carbamoyl derivatives tended to be more potent than the 5-carbamoyl ones. The 6-carbamoyl and 6-alkylamino derivatives were found to be more potent inhibitors against human type I enzyme than type II but on whole compounds were found not to be selective [212]. The non-steroidal o-hydroxyaniline (261; ONO- 3805) was a weak compound in vitro versus human 5␣-reductase II. It is a bi-substrate inhibitor in which the butanoic acid moiety is thought to be localized in the region of the phosphate group of NADPH and the lipophilic part could be orientated in the region of the steroidal C and D-ring, thus occupying the hydrophobic pocket of the enzyme. The fact that this compound acts as non- competitive inhibitor (versus T) and not as uncompetitive one, supports this hypothesis [173,213,214]. This prompted Pﬁzer to A series of indoline and aniline derivatives have also been prepare the derivatives of 261 and subsequently C-3 acylindole synthesized so as to inhibit both human and rat prostatic (367) was prepared which had improved potency versus both 5␣-reductase. Among the indoline series, 3-chloro-4-{[1-(4- human 5␣-reductase enzymes. The benzodioxolane (368) adopts phenoxybenzyl)indolin-5-yl]oxy}benzoic acid (363; YM-36117) a similar minimum conformation to the ether (367) and proved was found to be the most potent inhibitor against human enzyme to be a potent dual inhibitor of both 5␣-reductase enzymes. having an IC50 value of 5.3 and 46 nM against the rat enzyme while In common with the steroidal carboxylic acid inhibitors, these in aniline series, 3-chloro-4-{4-[N-(4-phenoxybenzyl)amino] phe- compounds require the carboxylic acid moiety for potency and noxy}benzoic acid (364) turned out to be most potent inhibitor the 3-acylindole motif was found to be crucial for dual activity with an IC50 against human and rat enzyme as 10 and 5.5 nM, presumably by allowing access to both the conformations 369 respectively [210]. and 370. The corresponding 2-methyl analog 371 which adopts conformation 370 due to the presence of methyl group on the S. Aggarwal et al. / Steroids 75 (2010) 109–153 147

Table 37 In vitro screening of compounds 367–371 against human 5␣-reductase I and II and rat 5␣-reductase.

Compounds Rat 5␣-reductase, Human type I Human type II

IC50 (nM) 5␣-reductase, IC50 5␣-reductase, IC50 (nM) (nM)

367 1404 368 92523 371 588 10 6300 ONO-3805 (225) 1.7 – 256 indole ring was found to be a selective inhibitor of 5␣-reductase I(Table 37) [215]. FK-143 (372) 4-[3-[3-[bis (4-isobutylphenyl) methyl amino] benzoyl]-lH-indol-l-yl] butyric acid was disclosed 34. Miscellaneous non-steroidal inhibitors by Sawada et al. as a potent dual inhibitor of both human 5␣- reductase isozymes. It inhibited in vitro human and rat prostatic In search of novel non-steroidal mimics of steroidal inhibitors of 5␣-reductase, 4-(2-phenylethyl)cyclohex-1-ene carboxylic acids 5␣-reductase in a dose-dependent manner with an IC50 of 1.9 and 4.2 nM, respectively, in a non-competitive fashion while in were synthesized with different substituents in para position of vivo showed potent inhibitory activity against castrated young rat the phenyl ring such as N,N-diisopropylcarbamoyl (373), phenyl, model. This compound can be a potential drug for the treatment of benign prostatic hyperplasia [216–218]. 148 S. Aggarwal et al. / Steroids 75 (2010) 109–153 phenoxy, etc. They turned out to be good inhibitors of the human Due to the excellent estrogen receptor binding afﬁnity of a prostatic 5␣-reductase isozyme II with 373 being the most potent series of 2,6-disubstituted 4-hydroxy-4-hydroxymethyl biphenyl one (IC50 = 760 nM) [219]. derivatives Lesuisse et al. designed various biphenyls as surrogates of the steroidal backbone. They hypothesized that by introducing appropriate substituents non-steroidal estrogens could be tai- lored into 5␣-reductase inhibitors. Two compounds (377 and 378) emerged as potent type II 5␣-reductase inhibitors with IC50 being 71 and 9.8 nM, respectively [221].

Fan et al. evaluated a series of umbelliferone (7- hydroxycoumarin) derivatives as inhibitors of 5␣-reductase I on LNCaP cells. The coumarin skeleton was considered as a mimetic of the proposed transition state of the natural substrate and as well as bioisostere of quinolin-2-one 1,1-dimethylallyloxycoumarin (374) showed potent inhibitory activity (IC50 = 1.3 ␮M) for the 5␣-reductase I. This was possibly a result of conformational effects of geminal dimethyl group. 8-Allyl-7-hydroxycoumarin (375) also Chen et al. evaluated isoﬂavonoids as potential non-steroidal exhibited potent inhibitory activity (IC = 0.99 ␮M) against 5␣- 50 inhibitors of rat 5␣-reductase by using the hypothetical phar- reductase I enzyme. Introduction of a carbonyl group at 7-position macophore of 5␣-reductase. They proposed that although these (376) resulted in only a slight increase in 5␣-reductase I inhibitory compounds (379–382) were inhibitors of rat 5␣-reductase in the activity (IC50 = 0.49 ␮M) [220].

range of 27–49 ␮M they could be evaluated as human 5␣-reductase inhibitors [222]. S. Aggarwal et al. / Steroids 75 (2010) 109–153 149

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