An overview on 5 -reductase inhibitors · b I. S. F College of Pharmacy, Ferozepur Road, Moga, Punjab 142001, India article info Article history: Received 13 July 2009 Received in

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Steroids 75 (2010) 109–153

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Steroids

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n overview on 5�-reductase inhibitors

aurabh Aggarwala, Suresh Tharejaa, Abhilasha Vermaa, Tilak Raj Bhardwaja,b, Manoj Kumara,∗

University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, IndiaI. S. F College of Pharmacy, Ferozepur Road, Moga, Punjab 142001, India

r t i c l e i n f o

rticle history:eceived 13 July 2009eceived in revised form 9 October 2009ccepted 20 October 2009vailable online 30 October 2009

eywords:�-Reductase inhibitorsndrogenszasteroidsestosteronePH�-Dihydrotestosterone

a b s t r a c t

Benign prostatic hyperplasia (BPH) is the noncancerous proliferation of the prostate gland associatedwith benign prostatic obstruction and lower urinary tract symptoms (LUTS) such as frequency, hesitancy,urgency, etc. Its prevalence increases with age affecting around 70% by the age of 70 years. High activity of5�-reductase enzyme in humans results in excessive dihydrotestosterone levels in peripheral tissues andhence suppression of androgen action by 5�-reductase inhibitors is a logical treatment for BPH as theyinhibit the conversion of testosterone to dihydrotestosterone. Finasteride (13) was the first steroidal 5�-reductase inhibitor approved by U.S. Food and Drug Administration (USFDA). In human it decreases theprostatic DHT level by 70–90% and reduces the prostatic size. Dutasteride (27) another related analoguehas been approved in 2002. Unlike Finasteride, Dutasteride is a competitive inhibitor of both 5�-reductasetype I and type II isozymes, reduced DHT levels >90% following 1 year of oral administration. A numberof classes of non-steroidal inhibitors of 5�-reductase have also been synthesized generally by removingone or more rings from the azasteroidal structure or by an early non-steroidal lead (ONO-3805) (261). Inthis review all categories of inhibitors of 5�-reductase have been covered.

© 2009 Elsevier Inc. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1102. Enzyme 5�-reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1103. Steroidal 5�-reductase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1114. 2- and 3-Azasteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1115. 4-Azasteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1126. 6-Azasteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1217. 7-Azasteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1248. 8-Azasteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1249. 9-Azasteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12410. 19-Nor-10-azasteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12411. 11-, 12a-, 13-Azasteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12512. 15- and 16-Azasteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12513. 17- and 17a-Aza-D-homosteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12514. Diazasteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12515. 11,13,15-Triazasteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12616. B,D-Dihomo-azasteroids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12617. Des-AB-azasteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12618. Steroidal 3-carboxylic/phosphonic/phosphinic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
19. Diazoketone steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20. 4-Substituted steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21. Steroidal oximes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22. Steroidal tetrahydrooxazin-2-ones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
∗ Corresponding author. Tel.: +91 172 2534115; fax: +91 172 2534112.E-mail addresses: [email protected] (S. Aggarwal), manoj [email protected] (M. Kum

039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.oi:10.1016/j.steroids.2009.10.005

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ar).

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110 S. Aggarwal et al. / Steroids 75 (2010) 109–153

23. 16-Substituted steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13024. 6-Methylene steroidal derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13025. Seco steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13126. Derivatives of natural substrate: pregnane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13127. Non-steroidal 5�-reductase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13528. Mimics of 4-azasteroids: benzo[f]quinolinones. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13529. Pyridones, quinolinones and piperidines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13630. Mimics of 6-azasteroids: benzo[c] quinolinones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13931. Mimics of 10-azasteroids: benzo[c] quinolizinones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13932. Non-steroidal aryl acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14133. Bisubstrate inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14634. Miscellaneous non-steroidal inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14735. Conclusion and future ahead . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149. . . . . .

1

osd(ppcloaumhTns(u[tcmaohaeofpmnacblpibtTrcttb

The proposed chemical mechanism of T (1) reduction to DHT (2)by 5�-reductase catalysis involves the formation of a binary com-plex between the enzyme and NADPH, followed by the formation ofa ternary complex with the substrate T. A delocalized carbocation is

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Benign prostatic hyperplasia (BPH) is the noncancerous growthf the prostate gland resulting due to over-proliferation of thetromal and glandular elements of the prostate [1]. It is causedue to the augmented levels of the androgen dihydrotestosteroneDHT). In BPH, microscopic foci within specific regions of therostate grows to form macroscopic nodules which eventually dis-lace the normal prostatic tissue and results into the uretheralompression. This compression resulting due to increased cell pro-iferation and/or impaired apoptosis causes physical enlargementf the prostate gland and is referred to as static component. Inddition dynamic component involves sympathetic nerve stim-lation causing contraction of prostatic and uretheral smoothuscle which results into outflow obstruction [2]. Despite several

ypotheses the molecular trigger for BPH remains unknown [3].he incidence of BPH is about 70% at 70 years of age and becomesearly universal with advancing age. Clinically, BPH causes a con-tellation of symptoms known as lower urinary tract symptomsLUTS). The hallmarks of the LUTS include frequency, hesitancy,rgency, nocturia, slow urinary stream and incomplete emptying4]. Earlier the choice of treatment in BPH was watchful waiting,ransurethral resection of the prostate (TURP) or open prostate-tomy but due to the invasive nature and potential side effectsany medical therapies have emerged involving the suppression of

ndrogen stimulation of prostatic growth [5]. These therapies delayr eliminate the requirement of surgery. 5�-Reductase enzymeas emerged as a target for the pharmaceutical treatment of BPHs abnormally high activity of the enzyme in humans results inxcessive DHT levels in peripheral tissues and hence suppressionf androgen action by 5�-reductase inhibitors is a logical treatmentor BPH [6]. A large number of molecules have been synthesized asotential 5�-reductase inhibitors over the years. Several analoguesay also act as androgen receptor antagonists by preventing the

atural ligands of the androgen receptor such as testosterone (T) (1)nd DHT (2) from binding to the receptor. Combination of these twoategories of inhibitors may provide effective androgen receptorlockage without undesirable side effects of castrate testosterone

evels on muscle and bone mass, energy level and libido which are ofarticular concern [7]. Some earlier reports have been there cover-

ng various aspects of 5�-reductase enzyme and inhibitors [8–10]ut a comprehensive review of each category and structural fea-ures required for 5�-reductase inhibitory activity were missing.his review is an attempt to cover all categories of inhibitors of 5�-
eductase with an aim to list the most potent compounds of eachategory along with the special structural requirements that ledo 5�-reductase inhibitory activity and in vitro data obtained fromhe evaluation of steroidal and non-steroidal compounds that haveeen tested as inhibitors of 5�-reductase. In particular IC50 and Ki
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

values for relevant compounds have been compared according tothe molecular class. The values given are not comparable acrossthe studies and in each comparison a standard taken in the studyis mentioned.

2. Enzyme 5�-reductase

A significant correlation between the androgens and prostate iswell known. Testicular androgens constitute the most importantmitogenic factor in vivo for the prostate [11]. Normal circulatinglevels of androgens are required for the maintenance of structuralfunction, growth and integrity of the prostate tissue. However,androgens have no direct effect on prostatic epithelial cells inculture [12]. Androgens enhance the production of many growthfactors in the prostate tissue in vivo through a complex cell tocell interaction involving both epithelial and stromal prostatic cells[13]. Androgen signalling cascade involves the synthesis of T (1) intestes and adrenal glands which gets peripherally converted to DHT(2). DHT formed gets transferred to the target tissues and binds tothe target receptor with consequent modulation of gene expression[14]. Both T and DHT bind to and activate the androgen receptor(AR), but DHT shows a higher affinity leading to different kineticprocesses. DHT dissociates from AR protein much more slowly thanits precursor. Therefore, at a given time ARs are occupied by DHTmuch more than by testosterone. T is converted to DHT by a steroid5�-reductase enzyme (3-oxo-steroid-4-ene dehydrogenase {E.C.1.3.99.5}) which is a system of two membrane bound nicotinamidedinucleotide phosphate (NADPH) dependent enzymes at the levelof prostatic stromal and basal cells. This has led to the develop-ment of steroidal and non-steroidal 5�-reductase inhibitors as theyinhibit the conversion of T (1) to DHT (2) as shown in Fig. 1 [15–17].Thus 5�-reductase dictates the cellular availability of DHT to pro-static epithelial cells and consequently modulates its growth.

Fig. 1. Site of action of 5�-reductase inhibitors.

S. Aggarwal et al. / Steroids 75 (2010) 109–153 111

n of 3

faEtrttilemciitu

att5cca

Npis

fh(amhmttlaeclma

wwtwts

Fig. 2. 5�-Reductio

ormed due to the activation of the enone system by a strong inter-ction with an electrophilic residue (E+) present in the active site.nolate of DHT is formed by the direct hydride transfer from NADPHo the � face of the delocalized carbocation leading to a selectiveeduction at C-5. This enolate which is coordinated with NADP+ onhe � face, is attacked by a proton on the �-face at C-4 giving theernary complex E-NADP+-DHT. Binary NADP+–enzyme complexs formed after departure of DHT and finally the release of NADP+

eaves the free enzyme for further catalytic cycles. Three differ-nt types of inhibitors could be conceived according to the kineticechanism of testosterone reduction: type A inhibitors which are

ompetitive with the cofactor (NADPH) and the substrate (T) andnteract with the free enzyme; type B inhibitors which are compet-tive with the substrate and fit the enzyme–NADPH complex, andype C inhibitors which fits the enzyme–NADP+ complex exhibitingncompetitive mechanism versus the substrate [8,18].

More potent inhibitors of steroid 5�-reductase have been foundmong the transition state analogues as molecules mimicking theransition state of the enzymatic processes exhibit a greater bindingo the enzyme and hence produce greater inhibition. The enzyme�-reductase binds the 3-keto-�4 steroids in such a way that thearbonyl group is brought into vicinity of a positively chargedentre on the enzyme whereby the conjugated ketone becomesctivated as shown in Fig. 2.

A hydride ion can then be transferred from the coenzymeADPH to the 5�-position of the steroid. The resulting enolate isrotonated at the axial 4�-position by the solvent and the product

s released. This evidence for protonation was based on the modeltudies with the Penicillium decumbens 5�-reductase enzyme [19].

Modern methods of molecular biology had assisted in identi-ying two types of 5�-reductase enzyme: type I and type II fromuman and rat prostatic complimentary deoxyribonucleic acidcDNA) libraries and the structures of both genes were elucidatedt the beginning of this decade [20,21]. The type I enzyme is not theajor species expressed in the prostate and is present mainly in the

air follicles and peripheral skin whereas type II 5�-reductase is theajor isozyme in genital tissues and a deletion in the gene leads

o male pseudohermaphroditism [22,23]. Type I enzyme is consti-utively expressed in the brain and in adulthood appears mainlyocalized in the myelin membranes and has a catabolic rather thann activating role in the brain while type II enzyme is transientlyxpressed in the prenatal period and in males its expression isontrolled by androgens and appears to be confined in the hypotha-amus and in the hippocampus after stress hence type II enzyme

ight participate in the perinatal differentiation of brain towardsmale pattern [24].

The two isozymes differ in the constitution of amino acids asell as molecular weight. The type I isozymes is active at pH 6–9
hile type II is active at pH 5.5. The two isozymes also differ in
he location of the gene structure while type I is located at 5p15hile type II is located at 2p22 although they had same gene struc-

ure [8,25]. The comparison of the properties of two isozymes isummarized in the Table 1:

-keto-�4 steroids.

More recently with the development of genome-wide geneexpression profile analyses a third type of 5�-reductase enzyme(type III) has been identified in hormone-refractory prostate can-cer cells (HRPC) [26]. This enzyme also converts T to DHT in HRPCcells in a similar way to type I enzyme and was found to be activeat pH 6.9 [27]. Type III isozyme has been recognized as a ubiquitousenzyme in mammals. Northern blot and real time RT-PCR analyseshave identified this enzyme in both androgen and non-androgentarget human tissues such as pancreas, brain, prostate cancer celllines, skin and adipose tissues [28].

Based on their structure 5�-reductase inhibitors are discussedbelow as steroidal and non-steroidal inhibitors.

3. Steroidal 5�-reductase inhibitors

As the only information available about the 5�-reductaseisozymes is their primary sequence estimated from c-DNAs thedesign of novel inhibitors is affected. Due to the unstable natureof enzyme during purification its crystal structure is not known.The first inhibitors have been therefore designed by modifying thestructure of natural substrates, including the substitution of onecarbon atom of the rings of the steroids by a heteroatom such asnitrogen thereby forming azasteroids. Singh and coworkers [29,30]as well as other groups have published comprehensive reviews onbiological activity of azasteroids [31]. Azasteroidal compounds hav-ing nitrogens at various positions have also been covered in thisreview. However, their 5�-reductase inhibitory activity has eithernot been done or they are devoid of activity. Some azasteroids havebeen found to be 5�-reductase inhibitors. In the following sectionazasteroidal inhibitors have been discussed depending upon theposition of nitrogen in the steroidal nucleus, i.e. nuclear azasteroids.

4. 2- and 3-Azasteroids

Although some of the 3-azasteroids were synthesized byDoorenbos and Wu [32] and Mazur [33] in the early 1960s butAnderson and Liao in 1968 reported for the first time that steroidalN-oxido-3-aza-1,3,5(10)-triene is a good inhibitor of enzyme 5�-reductase [34]. Haffner in 1994, reported the synthesis of somenovel 3-pyridyl-N-oxide steroids (3 and 4) [35] which mimic theenolate or enol like transition state of the enzyme–substrate com-plex.


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

Properties Type I 5�-reductase Type II 5�-reductase

Size 259 amino acids 245 amino acidsMolecular weight 29,462 Da 27,000 DaOptimal pH 6–8.5 5.0–5.5Biochemical properties Hydrophobic HydrophobicGene location SRD5A1, 5p15 SRD5A2, 2p23Gene properties 5 exons, 4 introns 5 exons, 4 intronsIn vitro inhibition by Finasteride Ki ≥ 300 nM Ki = 3–5 nMLocalization (in tissues) Sebaceous glands of the skin, sweat glands, dermal papilla cells

fibroblasts from all areas, epidermal keratinocytes, follicularkeratinocytes

Prostate, genital skin, epididymis, seminalvesicles

tional

t5

3s

filte

5

u1h(pwlis

Selectivity to the inhibitors Inhibitors with 4-methyl-4-aza func

a Ref. Li et al. [8].

N-Oxide steroids (3) and (4) were assayed against both type I andype II 5�-reductase and proved to be potent inhibitors of type II�-reductase with the Ki (�M) being 0.031 and 0.104, respectively.

In 2003, Robinson et al. reported the synthesis of various 2- and-azasteroidal derivatives (5–10) as effective and stable transitiontate 5�-reductase inhibitors [36].

All the synthesised 2- and 3-azasteroids (5–10) were evaluatedor human 5�-reductase inhibition. Amines (6 and 10) showed poornhibitory activity against both type I and type II isozymes whereasactams (5 and 9) displayed only marginal improvement againstype II isozymes. However, nitrones (7 and 8) showed significantnhancement in biological activity.

. 4-Azasteroids

4-Azasteroids is one of the extensively studied and clinicallysed classes of azasteroidal 5�-reductase inhibitors. Voigt et al. in970, screened a large number of steroids including 23 steroidalormones for their ability to inhibit the conversion of T (1) into DHT2) by a crude cell free enzyme system isolated from rat ventral
rostate [37]. In 1973, series of effective 5�-reductase inhibitorsere synthesized and evaluated. From the studies it was estab-
ished that the key structural requirements for the 5�-reductasenhibitory activity were presence of 4-en-3-one function and 17�-ide chain having one or more oxygen functionalities. Molecules

ity are very potent 4-Aza, 6-aza and charged 3-substituentsderivatives are highly selective

possessing these features act as competitive inhibitors of testos-terone 5�-reductase, therefore, all of them could be regarded as asubstrate of the enzyme 4-en-3-one steroids [38]. 4-Androsten-3-one-17-carboxylic acid (11) was identified as a potent inhibitor of5�-reductase.

It has been reported to be a competitive inhibitor of the enzyme
and showed 87.7% inhibition for the microsomal enzyme of humanskin. None of the compounds from this series could be shownto interfere with in vivo conversion of the dihydrotestosterone,because of their rapid conversion into the inactive 4,5-dihydro formby the enzyme.


AR of

aotaae3t

(eTtt[kacnt(

mc

ao15d3s

Fig. 3. Basic S

In search for a nonreducible inhibitor of 5�-reductase, Mercknd Co. in 1980 reported series of 4-azasteroids where C-4 of 3-xo-5�-steroids was replaced by nitrogen. The studies showedhat there was not only an increase in the 5�-reductase inhibitoryctivity but also retention of the in vivo activity [39,40]. Therefore,zasteroids were designed to mimic the putative enzyme-boundnolate intermediate by incorporating sp2-hybridized center at C-and C-4. Thus a lactam was introduced in the ring A of the steroids

o mimic the enol transition state of the enzyme–NADPH–substrate

E.NADPH.S) complex. Substitution at C-17 has been found tonhance potency by binding to a lipophilic pocket on the enzyme.hese competitive inhibitors strongly interact with the enzyme athe active site and on other hand unlike the substrate cannot be fur-her reduced to 5�-metabolites thus have in vivo inhibitory activity41]. The steroidal pharmacophore provides an anchor between theey A-ring lactam and the C-17 substituent while the former acts astransition state mimic of intermediate enolate, the latter signifi-

antly enhances potency via binding at a pocket largely lipophilic inature. The key 4-aza-3-oxo-5�-androstane pharmacophore andhe basic structure activity relationship (SAR) is outlined belowFig. 3) [42,43].

Taking into consideration that substitution at C-17�- could dra-atically affect the potency; a large number of modifications were

arried out to find potent inhibitors [44–46].4-MA {17�-N,N-diethylcarbamoyl-4-methyl-4-aza-5�-

ndrostan-3-one} (12) was found to be a potent dual inhibitorf both human 5�-reductase isozymes having IC50 value of
.9 nM against human 5�-reductase II and 1.7 nM against human�-reductase I, however, it was withdrawn from the clinicalevelopments due to hepatic toxicity and lack of selectivity over�-hydroxy steroid dehydrogenase enzyme [47–49]. Out of theeries its unsaturated analogue 17�-(N-tert-butylcarbamoyl)-4-
4-azasteroids.

aza-5�-androst-1-en-3-one, MK-906, Finasteride (13) was foundto be the best and extensively studied. Finasteride (13) was apotent inhibitor of 5�-reductase type II with only weak in vitroactivity versus 5�-reductase type I having IC50 value of 9.4 and410 nM, respectively. At clinical dose, 5 mg/day, it caused 65–80%lowering of plasma DHT levels [50]. Finasteride (13) was thefirst drug to be approved in U.S. for BPH. Long-term studies havedemonstrated that there is a sustained improvement in BPHdisease and reduction in the prostate specific antigen (PSA) level[51].

It was reported by Merck as well as Glaxo in 1996 that Finas-teride (13) and close analogues are mechanism-based inactivatorsof 5�-reductase II. Although it is accepted as an alternate substrateand is ultimately reduced to dihydrofinasteride (15), this pro-ceeds through an enzyme-bound NADP-dihydrofinasteride adduct.Initially it was believed that Finasteride (13) act as a transitionstate mimic whereby confirmation of the A-ring lactam closelymimics the enol form of transition state of 5�-reduced testos-terone but now it is understood that the most likely cause ofthe slow offset inhibition is rate-limiting hydride transfer fromNADPH to the �1-double bond of Finasteride (13). In the case ofthe 1,2-ene-containing Finasteride (13), reduction of C1 enablesthe nucleophilic attack of C2 on the nicotinamide C4. This aber-rant reduction results in the formation of lactam enolate whichis not positioned for efficient protonation by the enzyme. Insteadthe enolate is trapped by the electrophilic pyridinium cation of theNADP, yielding a covalent adduct to the cofactor and to the pro-tein (Fig. 4). This dihydrofinasteride–NADP adduct is a remarkablypotent bisubstrate analog inhibitor and it binds to the free enzyme
with a second-order rate constant equal to kcat/Km for turnover ofT (1) and has a dissociation constant Ki ≤ 1 × 10−13 M. Finasteride(13) is also a mechanism-based inhibitor of the human skin (typeI) isozyme, but it is processed with a much smaller second-orderrate constant, ki/Ki = 3 × 103 M−1 s−1, which attenuates its activ-


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

Table 2In vitro screening of compounds 20–27 against type I and type II human steroid5�-reductase.

Compounds Human 5�-reductasetype I, IC50 (nM)

Human 5�-reductasetype II, IC50 (nM)

20 20 0.221 350 24.622 >1000 25.223 120.2 0.424 5.6 <0.125 14.0 <0.126 8.1 0.2

it[

m3iwroac

Table 3In vitro screening of compounds 28–33 against human and rat prostatic 5�-reductase.

Compounds Human 5�-reductase,IC50 (nM)

Rat 5�-reductase, IC50

(nM)

28 41 8329 212 –Turosteride (30) 55 5331 381 22732 1218 161133 1553 1154

has resulted into a fruitful search of the potent dual azasteroidinhibitors (20–26).

Dutasteride (27) 2.4 0.5Finasteride (13) 52 <0.1

ty against this isozyme in vivo. Indeed, Merck has demonstratedhe presence of (14c) in the inhibited form of 5�-reductase type I52–54].

Weintraub et al. in 1985 reported 20-(hydroxymethyl)-4-ethyl-4-aza-2-oxa-5�-pregnan-3-ones and their corresponding

-thiones (16–19). These compounds were tested in vitro fornhibition of testosterone 5�-reductase and were found to be

eak inhibitors with Kis in the 10−7 range. It was argued thateplacement of C-2 in the steroid nucleus by oxygen in the casef 4-aza-3-oxo-steroids would convert it to a urethane (17) fromlactam (16), respectively, thereby enhancing the polarity at C-3

arbonyl, and its affinity for the enzyme active site [55].

4-MA (12) 28 37

Bakshi et al. reported a series of 4-aza-3-oxo-5�-androstene-l7�-N-aryl-carboxamides as dual inhibitors of human type I andtype II steroid 5�-reductases [56]. Some of these compoundswere found to be potent inhibitors of both isozymes. Variation ofthe C-17 amide substituent on the 4-aza-3-androstane skeleton

eroids 75 (2010) 109–153 115

ph(sDttiadlu(rsimdIatu

S. Aggarwal et al. / St

Dutasteride (GG745), 17�-N-{2,5-bis(trifluoromethyl)-henyl)}-3-oxo-4-aza-5�-androst-1-ene-17-carboxamide (27)ad emerged as the most potent dual inhibitor from this groupTable 2) [57]. It has been approved by U.S. FDA in 2002, for theymptomatic treatment of BPH [58,59]. Unlike Finasteride (13),utasteride (27) is a competitive inhibitor of both 5�-reductase

ype I and 5�-reductase type II isozymes, reduced dihydrotestos-erone levels >90% following one year oral administration [60]. Its also a time dependent inhibitor as Finasteride (13) and it forms

stable complex with a slow rate of dissociation constant andoes not bind to the androgen receptor [61]. By reducing DHT

evel, it reduces the size of enlarged prostate, so improving therinary flow rate. It is about 60 times more potent than Finasteride13) and has been shown to decrease the risk of acute urinaryetention and BPH related surgery [62–64]. This greater degree ofuppression of serum DHT has been found to correlate with thentraprostatic DHT suppression. Dual inhibition of 5�-reductase is

ore beneficial than selective type II inhibition as dual inhibitionoes not allow the escape of DHT which can formed through typemediated synthesis thus providing greater efficacy as DHT levels
re suppressed to a great extent. Long-term studies have shownhat Dutasteride (27) a dual inhibitor is well tolerated during dailyse for up to 2 years. It had a tolerability profile comparable to that
of placebo with the exception of a modestly elevated incidenceof impotence and decreased libido compared with placebo. AlsoDutasteride did not clinically significantly impact bone metabolismmarkers, bone mineral density or lipid levels [65].

In a programme aimed at searching for novel 5�-reductaseinhibitors a series of C-17-acylurea-substituted 4-azasteroids(28–33) (Table 3) were synthesized by Di Salle et al. in early 1990sexploiting the tolerance of functionality at this position. Signifi-cantly greater potency was found with the derivative containingC-4 methyl group and a saturated A-ring [66].

116 S. Aggarwal et al. / Steroids

Table 4In vitro screening of compounds 38–43 against type I and type II human steroid5�-reductase.

Compounds IC50 (nM) or % inhibition at 100nM (given in parenthesis)

Human 5�-reductasetype I (transfected 293cells)

Human 5�-reductasetype II (transfectedSW-13 cells)

38 27.3 ± 3.4 (20 ± 3.1)39 5.3 ± 1.1 (46.3 ± 1.3)40 24.2 ± 1 (4.9 ± 0.2)41 1.3 ± 0.64 (56.3 ± 4.5)42 31.5 ± 6.0 (48.7 ± 4.2)43 11.5 ± 1.9 (39.7 ± 0.4)Finasteride (13) 262 ± 43.2 8.5 ± 0.4

Table 5In 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 >10045 0.91 ± 0.236 >10046 2.19 ± 0.476 >10047 2.35 ± 0.421 >10048 9.57 ± 1.745 14 ± 1.1149 16.9 ± 3.911 18.4 ± 1.541Finasteride (13) 26.3 ± 4.784 4.53 ± 0.96



50 1.77 ± 0.343 1000 > IC50 > 10051 2.42 ± 0.409 1000

(ti

aa

alkyl side chain of 4–5 carbon atoms. N-Amyl substituted 17�-formamide (45) was found to be one of the most promising inhibitorof 5�-reductase type I while N-heptyl (48) and N-octyl (49) showeddual inhibition of both isozymes of 5�-reductase (Table 5).

52 2.93 ± 2.158 3.75 ± 1.97753 10.5 ± 2.739 58254 5.44 ± 1.067 1000 > IC50 > 100Finasteride (13) 26.3 ± 4.784 4.53 ± 0.96

One of the most potent compound of this group, Turosteride30), a close analogue of 4-MA (12), but unlike 4-MA was foundo be devoid of binding at the rat androgen receptor and a weaknhibitor of 3�-hydroxy steroid dehydrogenase [67].

Other azasteroids which retained 5�-reductase inhibitoryctivity are 2-substituted (34), A-homo- (35) and 19-nor- (36)nalogues [41].

75 (2010) 109–153

Observation that selectivity of inhibitors can be increasedagainst type I isozyme by making correct choice of hydrophobicsubstituent at C-17 position led to development of various 17�-(N-ureylene-N,N′-disubstituted)-4-methyl-4-aza-3-one 5�-reductasederivatives (Table 4) (38–43) as 5�-reductase inhibitors as theyhave potent selectivity against 5�-reductase type I enzyme. Aza-steroids with N-cyclopropyl ring exhibit potent inhibitory activityagainst type I 5�-reductase. Increase in the chain length from N′-ethyl to N′-butyl the compound showed strong inhibitory activitywhile branching of alkyl chain decreased potency of compoundsand introduction of 1,2-double bond significantly reduced theactivity. Replacement of N′-alkyl chain with phenyl moiety gavethe most active compound (41) of the series [68].

From the studies that 17�-carboxamides at C-17 positionhave pronounced activity of 5�-reductase and possess andro-gen receptor activities, a number of 17�-(N-alkyl/aryl formamido)(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-steroids and were evaluated as 5�-reductase inhibitors by Li et al.(Tables 5 and 6). Structure activity relationship indicated that 5�-reductase type I enzyme has preference for N-substituted linear

eroids 75 (2010) 109–153 117

dh

Prptir

e


Compounds IC50 (nM)

Type 1 Type 2

55 1.7 21856 5.7 33057 1.6 29858 2.0 12559 0.6 14760 8.4 5.4MK-386 (61) 0.9 154Finasteride (13) 52 <0.1

Table 8In 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)Finasteride 51(6) 32(5)

a Number of assays in parentheses.


Similarly in series of 17�-[(N-alkyl/aryl) alkyl) arylamido]erivatives (Table 6) exhibited highly potent inhibitory activity foruman 5�-reductase type I [69].

Various 7�-substituted derivatives have also been prepared.reliminary screening of the compounds as inhibitors of 5�-eductase from human scalp and prostate revealed that theresence of 7�-methyl substitution in ring B, presence of choles-erol type side chain at C-17 and ketone functionalities at C-3n 4-azasteroids resulted in potent selective inhibitor against 5�-eductase type I [70].

4,7�-Dimethyl-4-aza-5�-cholestan-3-one (MK 386) (61)merged as one of the most potent inhibitor of type I 5�-reductase

(Table 7) [71].

During 1995, some 17�-hydroxy-17�-(�-hydroxy/haloalkyn-l′-yl)-4-methyl-4-aza-3-oxo-5�-androst-1-ene-3-ones were syn-thesised by Li et al. and their antiandrogenic activity wasreported [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 investi-gated FCE 27837 (N-[1,1,1-trifluoro-2-oxobut-3-yl]-3-oxo-4-aza-5�-androst-1-ene-17�-carboxamide) (62), for its endocrinologicalproperties in comparison with those of Finasteride (13) (Table 8)[74].


Table 9In vitro screening of compounds 63–66 against human steroid 5�-reductase.

Compounds Human 5�-reductase inhibition Ki (nM)

6666

Lrw


Compounds Human 5�-reductase inhibition Ki (nM)

67 0.568 2.369 3.2


Compounds Human 5�-reductase, IC50 (nM)

777

3 2.64 1.85 2.26 5.1

In order to have specific and dual inhibitors of 5�-reductaseabrie and associates synthesized several steroids having lactam ining A and substitution at 17� position. Several of the compoundsere found active (Tables 9 and 10) [75].

0 161 82 14

Panzeri et al. reported the syntheses of several 17�-substituted 4-aza-5�-androstan-3-one carboxamides with unsatu-ration between C-1 and C-2 (70–72). These were found to be highlypotent against human 5�-reductase enzyme (Table 11) [76].


Table 12In vitro screening of compound 73 against human steroid 5�-reductase.


73 36 ± 9 3.3 ± 1.2Finasteride (13) 470 ± 41 8.5 ± 1.2

Table 13In vitro screening of compound 74 against rat and human steroid 5�-reductase.

Compounds IC50 (nM)

Rat prostate 5�-reductase Human prostate 5�-reductase

74 36 262Finasteride (13) 11 18

(5i7

(catr

14c1Fde


Compounds Rat 5�-reductase%inhibition at10−8 M

Human 5�-reductase relativeinhibitory potency to MK-906(MK-906 = 1)

75 50 0.5576 74 1.677 74 2.978 39 <0.179 33 1.0Finasteride (13) 28 1.0


Compounds IC50 (nM)

Type I Type II

80 13 0.281 420 2082 30 21083 120 5084 410 1585 5 11Finasteride (13) 52 <0.1


Compounds IC50 (nM)

Type I Type II

86 30 21087 6 1088 2 789 6 20Finasteride (13) 52 <0.1

In 1996, Giudici et al. reported the synthesis of FCE 2826073) [(22R,S)-N-(1,1,1-trifluoro-2-phenylprop-2-yl)-3-oxo-4-aza-�-androst-1-ene-17�-carboxamide] (Table 12) as a potent dual

nhibitor of both 5�-reductase isozymes and it was found to cause4% reduction in the DHT levels [77].

CIBA-GEIGY Ltd. reported the synthesis of CGP53153N-(2-cyano-2-propyl)-3-oxo-4-aza-5�-androst-1-ene-17�-arboxamide) (74) (Table 13), a novel inhibitor of 5�-reductasend structurally related to Finasteride (13) was found to be 10imes more potent than Finasteride in reducing prostate weight ofat [78].

In 1996, Ishibashi et al. reported the synthesis of various1�-acetoxy, 11�-hydroxy, 11�-hydroxy and 11-oxo substituted-aza-5�-androstane analogues (75–79) with a diphenylmethyl-arbamoyl moiety at C-17 and their evaluation. Compounds with an1�-hydroxy or 11-oxo showed inhibitory activities comparable toinasteride (13). The 4-methyl 11�-hydroxy-4-aza-5�-androstaneerivative (77) was found to be most potent against rat and human
nzyme and more active than Finasteride (13) (Table 14) [79].
Merck in 1997 reported the synthesis of several 4-aza 5�-androstan-3-one 17�-(N-substituted carboxamides) (80–85) aspotent human type II 5�-reductase inhibitors. From the studies itwas indicated that the 17-amide N substituent included aromaticresidue potent dual inhibitors of type I and type II 5�-reductasewere obtained (Table 15).

1 eroids 75 (2010) 109–153

htTsWtef[

1atrv

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

Table 17In vitro screening of compound 90 against human steroid 5�-reductase.a

Compounds Type I Type II

90 3.9 ± 0.1 1.8 ± 0.3Finasteride (13) 313 ± 74 11.3 ± 2.6

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


Compounds Type I 5�-reductase,IC50 (nM)

Type II5�-reductase, IC50

(nM)

94 750 1.595 180 2.396 51 997 97 2.1

20 S. Aggarwal et al. / St

The addition of N4-methyl substituent in A-ring increasesuman androgen receptor affinity while addition of unsaturationo the A-ring (�1) increased human androgen receptor binding.he unsubstituted carbanilides in the �1-N4-methyl series showedome selectivity for type I 5�-reductase over type II enzyme.

hereas addition of aryl substitution at the 2-position increasedype II 5�-reductase binding, thus providing dual inhibitors withxcellent human androgen receptor binding. Compound (87) wasound to be the most potent inhibitor from this series (Table 16)80].

In 1998, Salle et al. reported synthesis of a novel compound PNU57706 [N-(1,1,1,3,3,3-hexafluorophenylpropyl)-3-oxo-4-aza-5�-ndrost-1-ene-17�-carboxamide] (90) as a potent dual type I andype II 5�-reductase inhibitor. PNU 157706 (90) was found toeduce prostate weight 16-fold than Finasteride (13) while the ED50alues being 0.12 and 1.9 mg/kg/day, respectively (Table 17) [81].

Fig. 5. SAR of 6-azasteroids.

98 40 3.999 1,300 5.7100 14,000 1.8101 3,500 3.4Finasteride (13) 150 0.18

eroids 75 (2010) 109–153 121

mnIv

6

b

TI


Menzenbach et al. reported the synthesis of several 17-ethylene-4-azasteroids as inhibitors of 5�-reductase. Azaestra-

one II (93) was found to be inhibitor of 5�-reductase withC50 = 34 × 10−10 (for prostate) and IC50 = 25 × 10−10 (for seminalesicle) [83].

. 6-Azasteroids

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

able 19n vitro screening of compounds 102–114 against human steroid 5�-reductase.


Type II 5�-reductase,IC50 (nM)

102 12 1.4103 9 <0.10104 4.5 <0.10105 30 <0.10106 150 3.2107 3.6 <0.10108 6.9 <0.10109 20 0.16110 20 0.12111 12 <0.10112 20 0.40113 1.0 <0.10114 4.0 <0.10Finasteride (13) 150 0.18

tural and charge polarization features of the transition state for theenzyme catalyzed transfer of hydride from NADPH to testosterone.The higher reduction potential of ketoenamine compared to that of�,�-unsaturated ketone prevents these compounds from acting assubstrates for 5�-reductase and they show slow offset inhibitioninstead of irreversible as shown by 4-azasteroids [53]. Structureactivity relationship has also been reported by Frye and associatesat 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 thestructure activity relationship of A- and B-rings versus type I andtype II 5�-reductase (Table 18).

Methylation at N-6 (95) and substitution of C-4 with smalllipophilic groups such as Cl (96), Br (97) and CH3 (98) increases typeI 5�-reductase activity selectivity 4-fold while type I 5�-reductaseactivity was decreased by unsaturation (99), 1,2 cycloproponation
(100) and C-2-methylation (101).
By careful optimization of the C-17 substituent along with com-bining A- and B-ring substitutions, potent dual inhibitors of bothisozymes of 5� reductase were obtained (102–114) (Table 19) [84].

1 eroids 75 (2010) 109–153

roeofCegtgt


It was found that optimizing C-17 group resulted in 5�-eductase type I inhibitory activity in 103 with 5–7-fold increasef activity in 105. Swapping a methyl ester (106) for an admantylster (108) provides selectivity towards 5�-reductase I. Preparationf analogues of 103 resulted in compounds (109–112) which wereound to be 16–200-fold selectivity towards type I 5�-reductase.ompounds (103, 111, 113 and 114) having ketone at C-17 provedxtremely potent inhibitors of type I 5�-reductase. Out of the
roup, compound (105) demonstrated efficacy equivalent to Finas-eride (13) in a castrated rat model of DHT dependent prostaterowth. In general, large lipophilic groups at C-17 provides selec-ivity against 5�-reductase I.
A variety of C-17 amide-substituted 6-aza-androst-4-en-3-oneswere prepared and evaluated against human type I and type IIsteroid 5�-reductase in order to optimize potency versus bothisozymes of 5�-reductase. Out of the study two series of potent andselective C-17 amides were discovered, 2,5-disubstituted anilidesand (arylcycloalky1) amides (115–121) (Table 20). Evaluation ofsome optimal compounds from this series in a chronic castratedrat model of 5�-reductase inhibitor induced prostate involution,and pharmacokinetic measurements identified compounds (117,118, 120 and 121) with good in vivo efficacy and half-life in the dog[85].

eroids 75 (2010) 109–153 123

ai

dw

Asp

tpUlar(t




115 4.2 <0.1116 4.6 <0.1117 8.8 <0.1118 1.3 <0.1

Rahier and Taton have reported synthesis of several novel 6-aza-B-homosteroids but they were not tested for 5�-reductaseinhibitory activity [91]. Later in 2000 and 2001, Wenge et al.[92] and Xie et al. described the synthesis of 6-azasteroids as


B-Homologated analogue of 17�-N, N′-diethylcarboxy-6-aza-ndrost-4-en-3-one (122) has also been found to be potentnhibitor of 5�-reductase inhibitor with IC50 = 318 nM [86].

Bergmann et al. synthesised 7-substituted �-4-6-azasteroiderivatives (123) as 5�-reductase inhibitors [87]. But activity dataas not reported for these compounds.

R1 = H or CH3; R2 = CH3; R3 = hydrogen, Alk-R4, X-Alk, C1-6-X-lk, XCO-Alk, Co-Ar, CO-NH-Ar, CO-NH-Het, etc., where Alk is C1–12traight or branched alkyl, Ar is phenyl, X is O, N or S, Het isiperidinyl, piperizinyl, pirrolidinyl, pyrrolyl, etc.

6-Azacholesten-3-ones (Table 21) were assayed against bothype I and type II 5�-reductase by Haffner [88]. All three com-ounds were found to be potent dual inhibitors of 5�-reductase.nlike the 4-azasteroids the cholesterol side chain imparts very

ittle selectivity between type I and type II 5�-reductase. It waslso found that C-7 methyl group might provide a potent 5�-eductase I selective compound. The �-C-7 methyl diastereomer124) proved to be 7-fold more active 5�-reductase I inhibitorhan �-diastereomer (125).

119 4.0 <0.1120 6.8 <0.1121 0.6 <0.1Finasteride (13) 150 0.18

Fang and Sharp in 1996 synthesized several 6-azaandrostenonesof the general structure (127) as 5�-reductase inhibitors [89].

16�-Aryloxy, -alkoxy and heteroaryloxy 6-azasteroids of thegeneral formula as given below were synthesised by Aster et al. andthe compounds were found to be potent inhibitors of 5�-reductase[90]. Compound (128) was found to be potent inhibitor of human5�-reductase type I with IC50 in the range of 0.1–1000 nM.



Compounds Type I 5�-reductase ki (nM) Type II 5�-reductase ki (nM)

p[at

7

[dt

8

ar

9

ic

1

itGa

a45ts1st

Table 22In vitro screening of compounds 129–135 against human steroid 5�-reductase typeII.

Compounds IC50 (nM) FinasterideIC50 (nM)

IC50rel (nM)(IC50rel = IC50

compound/IC50

Finasteride)

129 4600 ± 2990 3.4 ± 2.2 1389 ± 1295129:130 = 5:1 4600 ± 960 4.1 ± 0.7 1123 ± 318131:132 = 22:1 2900 ± 1190 3 ± 1.4 981 ± 609133:134 = 9:1 37 ± 6.7 2.2 ± 0.4 16.9 ± 0.4133:134 = 3.5:1 150 ± 33 4.3 ± 1.5 34 ± 12135 460 ± 229 5.5 ± 2.1 83 ± 52

Table 23In vitro screening of compounds 129–135 against human steroid 5�-reductase typeI in DU-145 cells.

Compounds IC50 (nM) IC50rel (nM) Selectivity 5�-reductase II:5�-reductase I

124 0.8 7.9125 1 1.2126 1 2.3

otent phosphatidylinositol phosphalipase C (PI-PLC) inhibitors93]. Kasal et al. in 2005 reported an efficient synthesis of 6-za-allopregnanolone as neurosteroid analogues but not evaluatedhem for 5�-reductase inhibitory activity [94].

. 7-Azasteroids

Some 7-azasteroids were synthesized in early 1970s95]. Morzycki and Sicinski reported the synthesis of 6,7-iazacholestane derivatives but they were not evaluated forhe 5�-reductase inhibitory activity [96].

. 8-Azasteroids

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

. 9-Azasteroids

No work has been published on 9-aza steroids as 5�-reductasenhibitor although some fungicides have been known from thisategory [100].

0. 19-Nor-10-azasteroids

On the basis of the molecular model of active site for type IIsozyme and to increase the activity and selectivity of compoundsowards both 5�-reductase type I and 5�-reductase type II.uarna et al. synthesized a novel class of compounds 19-nor-10-zasteroids (Tables 22 and 23) [101].

Best results were obtained with 9:1 mixture of �9(11) (133)nd �8(9) (134) 17 �-(N-tert-butyl carbamoyl)-19-nor-10-aza--androsten-3-one as it was found to be a good inhibitor of�-reductase type I and 5�-reductase type II. The enamine struc-
ure of ring A of 10-aza-steroid (136) is analogous to that of theubstrate like transition state. The presence of N atom at position0 increases the nucleophilic character of the carbonyl group andtabilizes the carbocation intermediate (137) by delocalization athe positive charge.
129:130 = 5:1 263 ± 63 6.7 ± 2 1:17131:132 = 9:1 127 ± 12 2.8 ± 0.4 1:1135 1134 ± 288 24.4 ± 7 2.5:1

The inhibitory potency of these compounds depends on thepresence of bridgehead N-10 atom conjugated with 4-en-3-onemoiety in A-ring, unsaturation in C-ring and substituent at C-17 position. 19-nor-10-aza steroids have low transitional barrierenergy values and more flexible as compared to 6-aza or 4-azasteroids [102].

Some 10a-azasteroids were also synthesized from fusidic acidbut were not evaluated for 5�-reductase inhibitors [103]. Guarnaet al. also synthesized 17�-[N-(phenyl) methyl/phenyl-amido]
substituted 10-azasteroids. Unexpectedly, 5�-H compounds werefound more active than their 5�-H counterparts, with (138)(IC50 = 279 and 2000 nM toward isoenzymes I and II, respectively)and (139) (IC50 = 913 and 247 nM toward isoenzymes I and II,

eroids 75 (2010) 109–153 125

r[

1

ai

1

[io

1

s1anfb[

aaaooMd(oovad

Table 24In vitro screening of compounds 149–152 againsthuman steroid 5�-reductase.

Compounds IC50 (�M)

149 4


espectively) being the most potent compounds of the series104].

1. 11-, 12a-, 13-Azasteroids

Though many 11-azasteroids [105–107], 12a-azasteroids [108]nd 13-azasteroids [109] have been prepared but 5�-reductasenhibitory activities have not been reported.

2. 15- and 16-Azasteroids

Many 15-azasterols have been synthesized as antifungal agents110–112] but none of them have been evaluated for 5�-reductasenhibitory activity. 16-Azasteroids have been synthesized but nonef the compound has evolved as 5�-reductase inhibitor [113,114].

3. 17- and 17a-Aza-D-homosteroids

Regan and Hayes, in their exemplary work, have synthe-ized several 17- and 17a-aza-D-homosteroids from several7-ketosteroid oximes [115]. But 17a-azasteroids attracted morettention when chandonium diiodide was established as a potenteuromuscular blocker [116]. 17 and 17a-Azasteroids have been

ound to possess numerous biological activities like gamma aminoutyric acid (GABA) receptor antagonistic [117–119], antifungal120], antineoplastic, mutagenic [121,122] and anti-inflammatory

ctivity [123]. The most interesting aspect concerning 17-D-homo-zasteroids is the possibility of “inverted action” or “backbinding”s proposed by MacDonald et al. [124]. Their proposition was basedn the fact that the steroids have the potential to bind in tworientations in the active site of various metabolizing enzymes.arcus and Talalay [125] first reported that 3(17) �-hydroxysteroid

ehydrogenase converts both T (1) and dehydroepiandrosterone141) to androst-4-ene-3,17-dione (140) (Fig. 6a). Other examples
f enzyme inhibition by inverted steroids have also appeared. Thexiranes 143 and 144 were found to be active site-directed, irre-ersible inhibitors of 3-oxo-�5-steroid isomerase (Fig. 6b) [126]nd the bromoacetates 147 and 148 act as affinity labels for estra-iol 17�-dehydrogenase (Fig. 6c) [127]. Research on 5�-reductase
150 15151 12152 40

inhibitors has shown that steroids without side chains can bind toenzymes with the A-ring of the substance simulating the D-ring ofthe substrate, while the D-ring emulates the A-ring. This could leadto 17-D-homo-azasteroids exhibiting same mechanism of action as4-azasteroids. However, up to now, research has been concentratedon 4-azasteroids as 5�-reductase inhibitors and 17a-azasteroidsremained unexplored avenue as far as their 5�-reductase inhibitoryactivity is concerned. Some 17a-azasteroids (149–152) evaluatedfor 5�-reductase inhibitory activity are summarized in the Table 24[124].

14. Diazasteroids

Eberbach and coworkers reported in 1996 a novel access to4,13-diazasteroid derivatives but they were not evaluated for 5�-reductase activity [128]. In the same year Stuart et al. first reported4,17-diazasteroids as potential inhibitors of 5�-reductase. TheFinasteride 17-aza-isomer (153) proved to be potent inhibitorof 5�-reductase II although less active than Finasteride (13) andits congeners. 4-Methylation (154) lowered the inhibition of the5�-reductase II enzyme. Removal of �1(2) unsaturation led to the
formation of compound (155) that is dual inhibitor of 5�-reductasetype I and type II and 4-methylation of 155 led to the increase inactivity in 156. While compound with �5(6) (157) showed only amoderate inhibition of 5�-reductase II activity (Table 25) [129].


Fig. 6. (a) Action of 3(17) �-hydroxysteroid dehydrogenase, (b) action of 3-oxo-�5-steroid isomerase, and (c) action of estradiol 17�-dehydrogenase.

75 (2010) 109–153

8,13-Diaza steroids were also synthesized by Göndös et al. in1998 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 aggrega-tion activity and some derivatives exhibited positive action but no5�-reductase activity has been investigated in these categories ofsteroids [131].

16. B,D-Dihomo-azasteroids

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

17. Des-AB-azasteroids

Trehan et al. synthesized des-AB-azasteroids but 5�-reductaseactivity studies were not done [134].

Steroidal 5�-reductase inhibitors which are extranuclear, i.e. inwhich nitrogen is not the part of steroidal nucleus but forms partof the side chain or attached group have also been explored as 5�-reductase inhibitors, therefore are discussed next.




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)

a N,N-Diethyl-3-oxo-4-methyl-4-aza-5�-androstrane-17�-carboxamide (4-MA)was used as a standard reference.

S. Aggarwal et al. / Steroids

Table 26In vitro screening of compounds 158–166against human steroid 5�-reductase.

Compounds Ki,app (nM)

158 30159 7–18160 26161 7–12162 7163 30–36164 32

1

(eCCocaa[tAEta

At

165 35166 50

8. 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-boundnolate intermediate by incorporating sp2-hybridized centers at-3 and C-4 and, most critically, an anionic carboxylic acid at-3 as a charged replacement for the enolate oxyanion. Becausef presumably favorable electrostatic interaction between thearboxylate and the positively charged oxidized cofactor, thecrylate preferentially binds in a ternary complex with enzymend NADP+, which leads to the uncompetitive kinetic mechanism135–137]. Activity is enhanced in analogues possessing an addi-ional unsaturation at C-5 (162–167) along with �3-unsaturation.t C-17, diisopropyl (158) and pivalyl (163) amides were optimal.pristeride (SK&F 105657) (163) entered the clinical trials forreatment of BPH and is a potent inhibitor of 5�-reductase II whileweak inhibitor of 5�-reductase I.

A series of estratriene-3-carboxylic acids containing an aromatic-ring had also been synthesized (167–171) (Table 27) with struc-

ural variations in the C-2 and C-4 substituents, in the degrees of

Table 27In vitro screening of compounds 167–171against human steroid 5�-reductase.

Compounds Ki,app (nM)

167 20168 30169 10170 35171 36

75 (2010) 109–153 127

unsaturation in the B- and D-rings, and in the C-17 carboxamidealkyl groups. Despite lacking C-19 methyl group these compoundswere found to be potent inhibitors of 5�-reductase [138].

Nitro derivatives also showed interesting structure activityrelationship patterns compared to carboxylic acids, compound(172) was found to be potent competitive inhibitor by binding toE-NADPH complex [139]. The sulphonic acid (173) [140], phospho-nic acid (174) and phosphinic acid (175) also proved to be thepotent inhibitors of human 5�-reductase but not so of rat 5�-reductase [141]. The affinities of phosphonic acid are relativelyless than phosphinic acid derivatives because the increased bulkat the 3-substituent, leading to a steric intolerance for binding toenzyme. Overall 3-phosphosteroids were weaker inhibitors thantheir corresponding steroidal-3-carboxy steroids. The function of

the C-3-moiety is presumably to act as an H-bond acceptor from aresidue in the enzyme, which would normally donate a hydrogenbond to stabilize the enolate. Since the negatively charged groups(CO2

−) or isosteres of the carboxylate (NO2) best mimic thisinteraction it indicates that a pKa-matched H-bond with a Lys orArg donor may be operative. In addition, an interaction betweenthe negatively charged C-3-moiety and the positively chargedNADP+ cofactor after the enzyme has turned over substrate is
possible, especially if the cofactor lies directly under the A-ring ofthe steroidal skeleton in the transition state and mimics thereof.Compound 176 was not as active due to the presence of alcoholicgroup as it was not able to provide sufficient negative charge andhence was a weak inhibitor of rat and human 5�-reductase.

1 eroids 75 (2010) 109–153

1

rhihzit

sta

2

joa(

Table 28In vitro screening of compounds 180–183 against human 5�-reductase.

Compounds Human 5�-reductasetype I (transfected 293cells) (IC50 nM)

Human 5� reductasetype II (transfectedSW-13 cells) (IC50 nM)

180 2.9181 709 437182 >1000 192


9. Diazoketone steroids

The primary evidence of a dramatic increase in the affinity of 5�-eductase and an inhibitor with a 5-juncture of A-/B-ring and sp2

ybridization at the C-3 and C-4 positions was obtained from thenhibition with a mechanism-based inhibitor (5,20R)-4-diazo-21-ydroxy-20-methyl-pregn-6-en-3-one (177) (RMI-18,341). Dia-oketone (177) had been reported to be a potent time-dependentnhibitor with a Ki of 35 nM (time-dependency is considered indica-ive of irreversibility) [142].

A mechanism of inhibition was proposed that the protonationteps implicated in the normal enzymatic transformation activateshe diazoketone functionality to a diazonium ion that could furtherlkylate some nucleophilic residue at the active site [143].

0. 4-Substituted steroids

The observation that an excellent inhibitor possessed a con-ugated system (sp2–sp2–sp2) at C-3, C-4, and C-5 positionsf A-ring of steroids together with a lipophilic group at C-17,range of 4-substituted-3-oxo-4-androstene-17(-carboxamides

180–183) (Table 28) were prepared and compared with the

183 981 387Finasteride (13) 218 8.47

Finasteride (13). Out of these 4-cyano compounds were foundto be potent inhibitors of 5�-reductase type II enzyme andsubstitution with groups like thiol led to decreased activity.This series of compounds were also found to be potent andro-gen antagonists [144]. Fei et al. carried out the synthesis ofsome novel 4-trifluoromethylsteroids and proposed them as novel5�-reductase inhibitors. Out of the series 4-trifluoromethyl-N-(t-butyl)-4-androsten-17�-carboxamide (184) emerged as the mostpotent inhibitor being 4 times more active than Finasteride (13)

[145]. 4-Cyanoprogesterone (185) was also found to be a potentinhibitor of both rat and human 5�-reductase enzymes (IC50 val-ues = 0.045 and 0.050 �M, respectively). The mechanism of action of4-cyano steroidal inhibitor was assumed to be the transition state
inhibitor because on reduction by the enzyme compound wouldform a stable 5–3-enol that would remain tightly bound to theactive site [146,147].

eroids 75 (2010) 109–153 129

2

(cfIbp

ephieb(


1. Steroidal oximes

A number of pregnenolone (186–190) and progesterone191–194)-based steroids were synthesized bearing a oxime grouponnected directly or via a spacer to the steroidal D-ring, capable toorm a coordinate bond with haeme iron of enzyme 5�-reductase.n contrast to the pregnenolone derivatives which showed no inhi-ition of 5�-reductase isozyme I and II, progesterone derivativesossessed marked inhibition towards type II.

Inhibitory potency of synthesized compounds against targetnzyme using whole cell assay revealed that C-20 oxime (191) dis-layed strong inhibition against both isozymes (IC50 = 1.63 againstuman type I and 0.58 �M against human type II). Unsaturation

n ring D (192 and 193) in conjugation with oxime group furthernhances inhibitory activity. Almost complete loss of activity haseen found toward type I in the derivative with keto group at C-6194).

Transferring the oxime group from positions 20 to 21caused an increased selectivity toward type II isozyme. Z-21-Hydroxyiminopregn-4-en-3-one (195) was found to be a potentialinhibitor of the type II (IC50 = 1.95 �M against human type I and0.30 �M against human type II).

However, none of the compounds showed activity near to thatof the reference drug Finasteride (13) (IC50 values being 45 and3 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 steroidaltetrahydrooxazin-2-ones (196–201) containing heterocycles

1 eroids

i3cecrgSl

er

2

et5i1lpaOaTt1a

of its dienone group by electrophilic activation toward nucle-ophilic attack at the 6-methylene group is having structure (210)[153].


nvolving O and N heteroatoms at position 17� of androst-4-en--one, respectively, as 5�-reductase inhibitors. The IC50 values ofompounds vary between 270 and 600 nM. The relative inhibitoryffect of the unsubsituted N-phenyl compound 196 is 0.20. Con-erning the effects of substituents at position 4 of the phenyling in 196, the introduction of an ethyl (197) or ethoxy (199)roup resulted in a weak enhancement of 5�-reductase inhibition.ubstitution with halogens (200 and 201) or methoxy (198) causedowering of inhibition ability [149].

Steroid 5�-reductase inhibitors that do not contain any nitrogenither as part of ring or extranuclear but yet found to inhibit 5�-eductase are discussed next.

3. 16-Substituted steroids

A series of 16-methyl substituted derivatives of androst-4-ne and estr-4-ene originally prepared as antiandrogens, wereested for their inhibitory activity on rat and human prostatic�-reductase. The inhibitory activity data indicated that IC50

ncreases in sequence in derivatives bearing 16�-methyl (203),6�-methyl (204) and 16,16-dimethyl substituents (205). Acy-

ation of 17-hydroxy group significantly increases the inhibitoryotency (IC50 (207) = 4.8 nM, IC50 (206) = 23.5 nM in rat prostatend IC50 (207) = 0.52 nM, IC50 (206) = 0.62 nM in human prostate).verall, in human prostate homogenates IC50 varies between 0.6nd 120 �m while in rat prostate it ranges from 1.6 to 1000 �M.his shows enzyme of human prostate is more sensitive thanhat of rat prostate to methyl substituted compounds. Overall6-methyl steroids were found to be weak inhibitors both in ratnd human enzymes compared to the existing ones [150].

75 (2010) 109–153

Certain 19-nor analogues have also been synthesized in order toimprove the inhibitory activity in 16-methylated derivatives. TSAA-291 (16-ethyl-17�-hydroxy-4-estren-3-one) (208) was found to bethe first anti-androgen known to have dual action of competitiveinhibition of 5�-reductase activity and androgen receptor complexformation. It showed a Ki of 1400 nM to the purified nuclei from ratprostatic tissues [151].

24. 6-Methylene steroidal derivatives

2′,3′�-Tetrahydrofuran-2′-spiro-17-(6-methylene-4-androsten-3-one) (209; L612,710) is a potent time-dependentinhibitor which causes the highest percentage of inhibition (81%)of rat prostatic 5�-reductase enzyme. The structure activityrelationship showed that 3-oxo-4-ene functionally was essentialto the inhibitory activity and that substituents at C-17 influencedthe inhibitory potency. The presence of the C-19 methyl group wasnot essential to the activity. The A-ring appeared to interact withthe entire active site of the enzyme. Furthermore, the affinity of aninhibitor to the enzyme was greatly enhanced by the introductionof a methylene group at C-6 whereas activity was completely lostwith a large radical such as iodomethylene group at C-7. Thus aseries of 6-methylene steroids were prepared and examined asirreversible inhibitors of rat prostatic 5�-reductase [152]. Another6-methylene steroid that is potent inhibitor due to priming

eroids 75 (2010) 109–153 131

2

5fiiratnl[


5. Seco steroids

(4R)-5,10-Seco-estra-4,5-diene-3,10,17-trione (211) and (4R)-,10-seco-19-nor-pregna-4,5-diene-3,10,20-trione (212) wererst found to be non-competitive and possibly irreversible

nhibitors of epididymal 5�-reductase. Radiographic crystallog-aphy studies of both compounds showed that the conjugatedllenic 3-oxo-5,10-secosteriod (211) has a conformation similar tohat of the normal tetracyclic steroid dione. Both compounds wereon-competitive inhibitors of 5�-reductase and have an affinity

abel for the enzyme with Ki of 5470 and 980 nM, respectively154,155].

26. Derivatives of natural substrate: pregnane

As a consequence of the important observation that pro-gesterone and deoxycortisone inhibits the synthesis of dihy-drotestosterone by competing with 4-en-3-one function of thetestosterone for the 5�-reductase enzyme it led Voigt and cowork-ers to synthesize number of progesterone derivatives [37,38]. Thesatisfactory result of 4-cyano-progesterone (185) [146], which

possessed marked inhibitory activity for 5�-reductase enzyme,stimulated great deal of interest to synthesize various 4- and6-halo-progesterone analogs (213–217). These compounds werefound to be potent antiandrogenic in nature when tested againstgonadectomized hamster seminal vesicles and were also found tobe inhibitors of 5�-reductase [156–158].

1 eroids

ic4efahote

woSip(sCi


Bratoeff et al. evaluated the antiandrogenic and 5�-reductasenhibitory activity of various 16-phenyl substituted-D-homoompounds (218–219), 16-methyl substituted steroids (220–222),-bromo compound (223) without a methyl group at C-16 and thepoxy compounds (224–225). Compounds 219, 222 and 223 wereound to possess both antiandrogenic and 5�-reductase inhibitoryctivity better than the Finasteride (13) [158,159]. The trienonesaving a more coplanar structure reacts faster with the nucle-philic portion of the enzyme in a Michael type addition reactiono form an irreversible adduct with a concomitant inhibition of thenzyme 5�-reductase than the dienones.

A range of 4-bromo-17-substituted-4-pregnene-3,20 dionesere also synthesized and evaluated as 5�-reductase inhibitors

n gonadectomized hamster seminal vesicle and flank organs.mall diameter of the pigmented flank organ and great reductionn the weight of seminal vesicle has been found with the com-ounds having p-fluorobenzoyloxy (226) and p-chlorobenzoyloxy227) indicating that the presence of more electronegative sub-tituent at C-17 position (p-halosubstituted phenyl) and halogen at-4 enhances the antiandrogenic activity as well as 5�-reductase

nhibitory activity [160,161].

75 (2010) 109–153

Several new pregnane derivatives were also synthesized andevaluated by the conversion of [3H] T to [3H] DHT in Penicilliumcrustosum broths and the conversion of [1,2-14C] sodium acetateinto lipids. Compounds 228 and 229 were found out to be potent5�-reductase inhibitors as they inhibit conversion of T to DHT andalso decreased the incorporation of radiolabeled sodium acetateinto lipids of the flank organs [162].

eroids

itd5a

d5


Cabeza et al. also reported the 5�-reductase inhibitory activ-ty and the antiandrogenic effect of novel 16-bromo substitutedrienedione, 16� methyl substituted dienedione and the triene-ione (230–232). Compounds 230 and 231 were found to Exhibit�-reductase inhibitory activity higher than the commerciallyvailable Finasteride (13) [163].

The in vitro inhibitory activity of some novel progesteroneerivatives was also determined and they were evaluated as�-reductase inhibitors as well as antagonists for the androgen

75 (2010) 109–153 133

receptor. The appropriate homologues extended by a methylenegroup at C-6 (233–236) showed better activity due to the pres-ence of exocyclic double bond that can react faster with the enzymein a Michael type addition reaction than the corresponding endo-cyclic diene. Compounds 233 and 234 showed IC50 values of 19 and100 nM, respectively [164,165].

In compound 237 which is an epoxy compound the nucleophilicenzyme attacked the electrophilic center at C-6 and in this processinhibits the enzyme. Several 3-substituted 4-pregnene-6,20-dionederivatives (238–246) have been synthesized and were evaluatedfor antiandrogenic as well as 5�-reductase inhibitory activity. Thesynthesized steroids have an �,�-unsaturated carbonyl moietyin common. It was found that accessible electrophilic �-carbonof an �,�-unsaturated carbonyl moiety reacted very readily witha variety of nucleophiles to form Michael adducts. The first stepinvolved the formation of an enzyme-steroid activated complexand in a subsequent step the nucleophilic portion of the enzyme(amino group) attacked the conjugated double bond of the steroidin a Michael type addition reaction to form an irreversible adducts[166].

1 eroids 75 (2010) 109–153

gs22cspvga[

ttaF[

(aarhmCta[

6rca3at




262 6500 –


The IC50 values of the compounds were found to increase pro-ressively as the substituent on the phenyl group of the esteride chain at C-3 became more electropositive, i.e. compound41 has an IC50 value of 3.0 nM as compared to compound43 which has an IC50 value of 4.0 nM. On the other hand theompounds (244–246) having saturated 4-pregnene-6,20-dionekeleton for, e.g. 245 exhibited higher IC50 value (3.7 nM) as com-ared to the unsaturated ones (240–243) for, e.g. 241 with aalue of 3.0 nM. It was also found that the presence of halo-en substituents in ester moiety at C-3 as well as double bondt C-16 increased the binding affinity for the androgen receptor167].

Several new progesterone derivatives were also synthesized byhe same group having dienone moiety as reported earlier. Some ofhem (247–248) were found to have high 5�-reductase inhibitoryctivity like 248 showing an IC50 value of 0.5 nM as compared toinasteride (13) showing an IC50 value of 8.5 nM in the same study168,169].

Bratoeff et al. synthesized two novel steroidal carbamates249–250) which are the esters of carbamic acid with substituentst the amino and esters ends (NHRCOOR1) and proposed thems novel class of inhibitors for human and hamster steroid 5�-eductase. These compounds have a longer half-life and areydrolyzed slowly in the liver due to which they have a better phar-acological activity when compared to the conventional esters.

ompound 249 has got a similar IC50 value (10 nM) as that of Finas-eride (8.5 nM) (13) while compound 250 has a higher IC50 value ofbout 50 nM apparently due to the presence of large bromine atom170].

Working on similar lines some more novel 4,16-pregnadiene-,20-dione derivatives were synthesized and evaluated as 5�-eductase inhibitors. In this work, it has been demonstrated thatompounds containing chlorine (251), bromine (252), iodine (253)
toms, and (254); without any substituent in the ester moiety) at C-produce a significant decrease of the prostate weight in castrated
nimals treated with testosterone (1). Therefore, it was proposedhat the ester moiety at C-3 is functioning as a pharmacophore,

263 460 –264 30 –265 60 –

enriched by the presence of halogens in these steroidal derivativesleading to the increase in the inhibition of 5�-reductase enzymeas determined by the IC50 values. Compound 252 was found to bemost potent with an IC50 value of 1.8 nM while compounds 251and 253 showed values 14 and 10 nM, respectively, in comparisonto Finasteride (13) having value 8.5 nM. Compound 254 was not

that active when compared to halogenated compounds thusfurther demonstrating the need of a halogen atom in the ester sidechain [171].

Recently, Cabeza et al. synthesized several C-6 substituted andunsubstituted pregnane derivatives as potential 5�-reductaseinhibitors. It has been found that steroids that lack a chlorineatom in C-6 (255–257) exhibited a higher capacity for inhibition ofthe activity of 5�-reductase (IC50 in the range of 25–63 nM) thanthe compounds (258–260) having this atom (IC50 in the rangeof 920–990 nM in comparison to Finasteride (13) having an IC508.5 nM). The presence of bromine atom in C-6 of compound 261
however doesn’t affect the inhibitory activity of enzyme (IC50being 33 nM). Also, the presence of an ester moiety in C-17 �on the steroidal skeleton tends to increase the inhibition of theactivity of the enzyme [172].

eroids 75 (2010) 109–153 135

2

hsetftr(ipvabmtwstiai

2

prwsaTta


7. Non-steroidal 5�-reductase inhibitors

A number of classes of non-steroidal inhibitors of 5�-reductaseave now been identified. It was anticipated that the use of non-teroidal template can decrease the potential interaction with othernzyme or receptor of the steroidal endocrine system and can limithe complexity of target compound synthesis [173]. They have inact emerged either from the design of compounds mimic of azas-eroidal inhibitors, generally by the formal removing of one or moreings from the azasteroidal structure or by early non-steroidal leadONO-3805) (261) which was prepared as leukotriene synthesisnhibitor [174] or by high throughput screening. These com-ounds are generally thought to act all as competitive inhibitorss. testosterone with exception of the epristeride analogues whichre uncompetitive inhibitors. Non-steroidal inhibitors includeenzo[f]quinolinones, pyridones and quinolinones which wereimics of 4-azasteroid inhibitors. Benzo[c]quinolinones were syn-

hesized as mimics of 6-azasteroids while benzo[c]quinolizinonesere designed as mimics of 10-azasteroids. The most potent and

elective inhibitors of human type I 5�-reductase are found amonghese classes of compounds. Almost all the other non-steroidalnhibitors can be grouped as carboxylic acid (generally butanoiccid) derivatives which are thought to act as non-competitivenhibitors versus testosterone in analogy to ONO 3805 (261).

8. Mimics of 4-azasteroids: benzo[f]quinolinones

Benzo[f]quinolinones were the first non-steroidal inhibitorsrepared by the Lilly’s researchers. They were derived by theemoval of the D-ring from 4-azasteroids and replacing the C-ringith an aromatic one [175]. Most of these compounds are type I

elective, although dual inhibitors can be obtained if an appropri-te substitution is present at the position 8 on the aromatic ring.wo main classes of benzo[f]quinolinones have been described,he hexahydro derivatives (262–265), which have an unsaturationt positions 4a–10a, and the octahydro derivatives (266–271). In

general octahydro derivatives are more potent inhibitors than thecorresponding 4a–10a unsaturated compounds (Tables 29 and 30)and in both series the potency toward type I 5�-reductase increasesif an halogen atom is present at position 8 (in particular a Cl atom)and a methyl group at position 4; in fact the most potent inhibitorof the series is LY191704 (268) with IC50 = 8 nM (Table 30). Thismolecule has progressed into human clinical trials.

The quantitative structure activity relationship study of thesecompounds has focused on the effect of 8 substituent on the aro-matic ring, which can be accounted for by its lipophilic character[176]. They found that the optimum activity may reside in the prop-erty space around the chlorine substituent. The substitution of the8-Cl atom by a F (270) or a Br atom (271) decreased very slightly the
potency. Finally, several kinds of substituents, including complexaromatic groups, were introduced at the position 8 [177], and somepotent type I selective inhibitors such as compounds 272–274 wereprepared.

1 eroids 75 (2010) 109–153

2

(tIoot


9. Pyridones, quinolinones and piperidines

Abell et al. synthesized a number of tricyclic thiolactams275–276), aryl acid (277), bicyclic lactams (278–281) and bicyclichiolactam (282) and evaluated them in vitro as inhibitors of type
and type II steroid 5�-reductase (Table 31). Removal of twor more rings from 4-azasteroids resulted in a strong decreasef potency. The tricyclic thiolactams were found to be selectiveype I 5�-reductase inhibitors and in general were less active
than the corresponding lactams. The aryl acid 277 showed gooddual isozyme inhibitory properties with significantly enhancedtype II activity. Bicyclic lactams, in general, were found to be lessactive against type I 5�-reductase than the tricycles. For example,compounds (278–279), lacking the B and D steroidal rings, werepoor type I 5�-reductase inhibitors, with the highest potencyassociated to the presence of the Cl atom on the aromatic ring of279 [178]. A styryl (or azo) substituent dramatically enhances typeII activity (and indeed type I activity with the bicycles) (280–281)and provided dual inhibitors of type I and type II.





266 60 –267 17 –268 8 10,000269 11 –270 35 –

ftdr

271 35 –272 59 >10273 6 1,400274 6 1,340

Hartmann and coworkers synthesized pyridones of the general
ormula 283 and 284 where the B- and C-rings of the steroid sys-em have been replaced by an acyclic linker but these compoundsisplay relatively weak activity versus both the rat and human 5�-eductase isozymes (Ki > 20 �M) [179–181].
75 (2010) 109–153 137

Their poor potency however illustrates the need for both A- andB-rings to be present with the correct fusion pattern for good recog-nition at the enzyme active site. A series of 5-phenyl substituted1-methyl-2-pyridones have also been prepared and tested againsthuman and rat 5�-reductase type I and type II. Compound 285bearing bulky carboxamide substituents exhibited excellent 5�-reductase type II inhibitory activity with IC50 value of 10 �M [182].

McCarthy and coworkers have recently synthesized a series of4′-substituted 5-aryl pyridones along with corresponding 1-aryl-pyridone derivatives and tested them against 5�-reductase typeI and type II expressed in transfected human embryonic kidneycells to examine structure activity relationship for the 4′ positionin pyridones.

1 eroids 75 (2010) 109–153

4i4lTi[

2bIqcbNq

2NTpfleahwt

pn



Type II 5�-reductase,IC50 (nM) orpercentage inhibition

275 377 13.2% @ 40 �M276 183 21.6% @ 40 �M277 152 340278 2477 13.5% @ 40 �M


Weak inhibition was observed against the type I isozyme for′-N-substituted acetamide compounds (286–289) while potent

nhibition of type I isozyme was observed for compound 290 having′-benzoyl substituent and also for compounds (291–292) having

ong carbon chain tethers attached to the 4′-acetamide (Table 32).hus further proving that large hydrophobic groups are toleratedn a region of the active site not involved in the enzymatic reaction183].

Few quinolinone derivatives such as 6-substituted 1H-quinolin--ones (293–294) and 2-methoxy quinolines (295–296) have alsoeen synthesized. The most active inhibitor for the human type

I isozyme was 6-[4-(N,N-diisopropylcarbamoyl) phenyl]-1H-uinolin-2-one 293 having Ki 800 ± 85 nM, showing mostlyompetitive inhibitory patterns. A type I selective inhibitor coulde identified with 6-[4-(N,N-diisopropylcarbamoyl) phenyl]--methyl-quinolin-2-one 294 (IC50 = 510 nM) but 2-methoxyuinolines were not found to be active [184].

Hartmann and coworkers synthesized and evaluated a series of′-substituted 4-(4′-carboxy- or 4′-carboxymethylbenzylidene)--acylpiperidines as active steroid 5�-reductase type II inhibitors.hey synthesized several compounds from N-acyl-4-benzylidene-iperidine-4′-carboxylic acids. In the dicyclohexylacetyl series,uorination in the 2-position of the benzene nucleus (297),xchange of the carboxy group by a carboxymethyl moiety (298)nd combination of both structural modifications (299) led toighly active inhibitors of the human 5�-reductase type II isozymeith IC50 values of 297, 298 and 299 being 11, 6 and 7 nM, respec-

ively, in comparison to Finasteride (13) having value of 5 nM [185].

Earlier Hartmann et al. have reported a series of N-substitutediperidine-4-(benzylidine-4-carboxylic acids) (300–303) as potenton-steroidal dual inhibitors of 5�-reductase.

279 1690 12,350280 302 579281 107 617282 3360 14% @ 40 �M



Compounds Type I 5�-reductase(% inhibition at 10 �M)

Type II 5�-reductase(% inhibition at 10 �M)

286 8 –287 6 –288 6 –289 3 –

taiIahbto

(mcMfaeaos

3

wt

double bond enabling conjugation between carbonyl and nitrogenatom [189–191].

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 andype II, respectively) and 302 (IC50 = 0.54 and 0.69 �M for type Ind type II, respectively) displayed the best inhibition toward bothsozymes. Compound 301 showed a strong inhibition toward typeI human and rat enzyme (IC50 = 60 and 80 nM) but only a moderatectivity versus type I enzyme (IC50 approximately 10 �M for rat anduman enzyme). Compound 303 (IC50 in human type II enzymeeing 0.26 �M and in rat type II enzyme being 0.29 �M) was foundo be a moderate dual inhibitor probably due to higher flexibilityf the open ring substituent [186].

Methyl esters of N-(dicyclohexyl)acetyl-piperidine-4-benzylidene-4-carboxylic acid) (304) were designed and

onitored for dual inhibition toward type II isozyme in BPHell free preparation and for type I isozyme in DU 145 cells.ethyl esters, applied as hydrolytically stable precursor drugs to

acilitate cell permeation, will yield the corresponding carboxyliccids as type II inhibitors after hydrolysis in the target organ. Thesters themselves stable in human plasma and Caco-2 cells acts potent drug toward 5�-reductase type I. Thus, dual inhibitionf 5�-reductase type I and type II can be achieved by applying aingle parent compound [187].

0. Mimics of 6-azasteroids: benzo[c] quinolinones

On the basis of 6-aza-androst-4-en-3-one derivatives (Fig. 5) inhich a vinylogous amide was inserted into a steroid nucleus as a

ransition state mimic for conversion of T (1) to DHT (2) and Lilly’s

Table 33In vitro screening of compounds 308–317against recombinant 5�-reductase I expressedin CHO cells.

Compounds IC50 (nM)

308 5130 ± 130309 176 ± 17310 459 ± 118311 137 ± 58312 49 ± 19313 20 ± 8314 7.6 ± 0.9315 14.3 ± 5.9316 8.5 ± 2.1317 204 ± 49

75 (2010) 109–153 139

Benzoquinoline derivatives (267–268), novel phenanthridin-3-onederivatives (305–307) were synthesized having vinylogous amidepharmacophore.

Although compounds were found to be 5�-reductase type Iselective and poor inhibitors of 5�-reductase type II but overallthese compounds did not showed promising inhibitory activity.The potency of compounds was found to increased from 305(Ki � 10 �M) to 306 (Ki = 1.1 �M) and 307 (Ki = 0.92 �M) this wasdue to the presence of methyl group on the A-ring correspondingto the 4-Me of Eli Lilly inhibitors. This effect is due to the presenceof hydrophobic pocket in the active site of enzyme which is able toaccommodate a small alkyl group located at the position 4 of theA-ring of these tricyclic inhibitors [188].

31. Mimics of 10-azasteroids: benzo[c] quinolizinones

Guarna et al. had synthesized two series of benzo[c]quinolizin-3-ones as novel inhibitors of human 5�-reductase type I: 4aH-serieswith a double bond between the positions 1 and 2 (308–311) and1H-series with a double bond between the positions 4 and 4a(312–317). The efficacy and selectivity of these compoundshave been demonstrated on recombinant human 5�-reductasetype I expressed in CHO cells but they displayed very poor orno inhibition towards 5�-reductase type II (Table 33). Increasedactivity of the compounds of 1H-series than those of correspondinginhibitors of 4aH-series has been attributed to the presence of

Fig. 7. SAR for �4-benzo[c] quinolizin-3-ones.

1 eroids 75 (2010) 109–153

ac5iTca

atmiuitv

dpa1t

TIa


The presence of a methyl group at position 4 (311, 313, 314,nd 316) associated with a substituent at position 8, gave potentompounds in comparison with Finasteride (13) and the known�-reductase I selective inhibitor LY191704 (268). All compounds

nhibited the enzyme through a reversible competitive mechanism.he structure activity relationship carried out on this class espe-ially methyl group at positions 1, 4, 5, 6 and 8 can be summarizeds follows (Fig. 7) [191].

It was found that presence of substituent at position 8, eitherchlorine or methyl group, generally increased the potency of

he inhibitors. Thus compound 309 and 310 were potent wereore active than unsubstituted compound 308 in 4aH-series. Sim-

larly in 1H-series compounds 312 and 314 were more potent thannsubstituted compounds due to presence of 8-chlorine group. The

ntroduction of a methyl group at position 4 was found to increasehe potency in both series with effect being higher in 1H-series. Aery strong increase of potency is observed in 8-chloro-4-methyl

erivative 314 when compared to 4-methyl unsubstituted com-ound 312. The substitution with a methyl group at position 6 alsoffects potency in both series with effect being more prominent inH-series with compound 315 being more potent than unsubsti-uted 312. This effect of methyl substitution at position 6 seems

able 34n vitro screening of compounds 318 and 319 against recombinant 5�-reductase Ind II expressed in CHO cells.


Type II 5�-reductase, IC50 (nM)

318a 58 ± 2.1 No inhibition319 20,000 ± 400 No inhibtion

a Mixture of �6a(10a)/�10(10a) isomers in 10:1 ratio.

consistent with the observation that the introduction of the samegroup on the corresponding position 7 in 4-azasteroids increasedtheir 5�-reductase I selectivity [70]. Presence of methyl group atposition 5 found to decrease the potency of the compounds whileintroduction of methyl at position 1 as in compound 317 causedonly a slight decrease in activity when compared to unsubstitutedcompound 312.

In 2001, Guarna et al. studied the effect of C-ring modificationsin benzo[c]quinolizin-3-ones. They synthesized several octahydro-and decahydrobenzo[c]quinolizin-3-one derivatives containingpartially or fully saturated C-ring. These compounds were foundto be selective 5�-reductase I inhibitors. Benzo[c]quinolizin-3-oneinhibitor lacking the aromatic C-ring but with a double bond at6a–10a 318 displayed an inhibitory potency 345-fold higher thanthat of the corresponding 6a–10a saturated, trans-fused compound319 (Table 34) [192].

A 3D-QSAR model correlating the potency of the inhibitors withtheir physicochemical features using density functional theory(DFT) was also developed for a series of benzo[c]quinolizin-3-onesderivatives by adding two “non standard” variables (dipole momentand log P) to the classical electrostatic and steric comparativemolecular field analysis (CoMFA) fields [193].

With the aim to discover new dual non-steroidal inhibitors of5�-reductase I and II a series of benzo[c]quinolizin-3-ones deriva-tives (320–325) bearing diverse substituents at position 8 weresynthesized in 2005 [194]. They were tested towards 5�-reductase
I and II expressed by Chinese Hamster Ovary cells (CHO 1827 andCHO 1829), respectively. It was found out that most potent dualinhibitors were obtained when F atom was introduced on the phe-nol moiety of these esters. All compounds displayed inhibitiontowards 5�-reductase I in the range of 93–165 nM (Table 35). Com-

eroids 75 (2010) 109–153 141

ps

3

(pwop7csc

i

Table 35In vitro screening of compounds 320–325 against recombinant 5�-reductase I andII expressed in CHO cells.

Compounds Type I 5�-reductase (%inhibition at 10 �M)

Type II 5�-reductase (%inhibition at 10 �M)

320 102 553321 129 584322 93 119323 160 134324 138 166325 42 368

Table 36In vitro screening of compounds 326–330 against recombinant human 5�-reductaseI and II.

Compounds Type I 5�-reductase (Ki,app)(nM)

Type II 5�-reductase(Ki,app) (nM)

326 315 >10,000327 320 ∼2,500328 26 10,000


ound 322 was found to be the most potent dual inhibitor of theeries with IC50 values about 100 nM for both enzymes.

2. Non-steroidal aryl acids

Some novel 9,10-dihydrophenanthrene-2-carboxylic acids326–328) were prepared by formally removing D-ring fromarent androstene carboxylic acid inhibitors and contrary to them,ere found to be selective 5�-reductase I inhibitors. Introduction

f a bromine atom at position 7 in compound 328 gave the mostotent compound of the series. Substitution by chlorine at positionin compound 327 does not result in increase in potency as

ompared to unsubstituted compound 326. These compounds areupposed to interact with the positively charged enzyme–NADP+

omplex in an uncompetitive manner versus testosterone [195].

Moreover when double bond was introduced in the B-ring asn compound 329 (formally obtained by removing the D-ring from

329 1200 260330 1900 1,600

Epristeride 163) the selectivity toward 5�-reductase I was foundto be lost in favor of an increased potency towards 5�-reductase II(Table 36) [196].

On removal of two or more rings from the parent steroidal com-pounds several aryl acids mimics of steroidal carboxylic acids havebeen synthesized (331–336). Hartmann et al. synthesized severalN-substituted 4-(5-indolyl) benzoic acids but potent and selec-tive human 5�-reductase I inhibitors were not found from theseries. Only compound 331 with IC50 value of 67 nM against human5�-reductase I was the most potent inhibitor [197]. Similarly, com-pound 332 was found to possess IC50 value of 680 nM [198].


a(4a1f2

B- and C-rings was more tolerant of variation. Both the A-ringcarboxylic acid and C-ring are critical for activity. Compounds 337and 338 were found to be most potent among the series withKi,app being 10 and 5 nM, respectively. In indole series there was

Fig. 8. SAR of benzophenone series.

Igarashi et al. synthesized a new series of indole derivativess potent human prostatic 5�-reductase inhibitors. Compounds333–336) were found to be most potent among the series with-[(1-benzyl-1H-indol-5-yl) oxy]-3-chlorobenzoic acid 334 havingn IC50 value of 0.44 nM while 3-chloro-4-{1-(4-phenoxybenzoyl)-H-indol-5-yl]oxy}benzoic acid 335 showed inhibitory activitiesor both human and rat prostatic 5�-reductase with IC50 values of.1 and 73 nM, respectively [199].

Fig. 9. SAR of indole series.

Screening of aryl carboxylate versus the human 5�-reductaseisozymes by SmithKline-Beecham company led to the discoveryof two series of selective and potent 5�-reductase II non-steroidalinhibitors based on benzophenone and indolecarboxylic acidsskeleton. In benzophenone series of inhibitors the linker betweenthe A- and B-rings proved to be crucial whereas linker between

eroids

aisa(

va5


strong preference for substitution at the 5- or 6-position of thendole ring. Compounds 339 and 340 were most active among theeries with Ki,app being 10 and 40 nM, respectively The structurectivity relationships of both the series are summarized as belowFigs. 8 and 9) [43,200].

Takami et al. synthesized various indole derivatives witharied substituents on the �,�-unsaturated double bondnd evaluated them for activity to inhibit rat prostatic
�-reductase. Among the various derivatives they found
75 (2010) 109–153 143

that (Z)-4-{2-[[3-[1-(4,4′-difluorobenzhydryl)indol-5-yl]-2-pentenoyl]-amino]phenoxy}butyric acid (341,KF20405) was themost potent compound having activity about 20 times greaterthan Finasteride (13) and an IC50 value of 0.48 ± 0.086 nM [201].

A novel series of indole and benzimidazole derivatives werealso synthesized and evaluated for their inhibitory activityof 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 potentinhibitory activities with IC50 values of 9.6 ± 1.0 and 13 ± 1.5 nM,respectively [202].


a[aa(s

watcsit

Sawada et al. synthesized a novel series of indolizinebutyriccids with various benzoyl substituents.FK687 (344) (S)-4-[1-[4-[1-(4-isobutylphenyl) butyl] oxy] benzoyl] indolizin -3-yl] butyriccid displayed strongest in vitro inhibitory activity (IC50 = 4.6 nM)gainst the human enzyme and in vivo inhibitory activityIC50 = 1.7 nM) against the castrated young rat model among theeries [203].

In 2002, various N-substituted 4′-biphenyl-4-carboxylic acidsere synthesized by increasing the conformational flexibility using

n ether linker between the steroidal A–C-ring mimetics and were
ested against human and rat 5�-reductase type I and type II. Twoompounds were found to be most potent with compound 345howing an IC50 value of 60 nM while 346 showed an IC50 valuemproved by a factor of 5 from 1.9 to 0.38 �M in comparison withhe parent biphenyl compound 347 [204].
75 (2010) 109–153

Baston et al. synthesized several 3,4-dihydro-naphthalene-2-carboxylic acids and evaluated them for 5�-reductase inhibitoryactivity. The most active inhibitors were 6-[3-(N,N-dicyclohexylaminocarbonyl) phenyl]-3,4-dihydro-naphthalene-2-carboxylicacid (348) (IC50 = 0.75 �M, human type II; IC50 = 0.81 �M, humantype I) and 6-[4-(N,N-diisopropylamino-carbonyl) phenyl]naphthalene-2-carboxylic acid (349) (IC50 = 0.2 �M, humantype II). The latter compound was shown to deactivate the enzymein an uncompetitive manner (Ki = 90 nM; Km, T = 0.8–1.0 �M)similar to the steroidal inhibitor Epristeride (163) [205].

Novel substituted benzoyl benzoic acids and phenylacetic acidswere synthesized by Salem et al. based on the template structure338 and were evaluated for the inhibition of rat and human steroid5�-reductase isozymes I and II. The phenylacetic acid derivativeswere more potent than the analogous benzoic acids. Bromination inthe 4-position of the phenoxy moiety led to the strongest inhibitorof the series against human 5�-reductase II (352; IC50 = 5 nM),which was equipotent to Finasteride (13) while compounds 350(IC50 = 23 nM) and 351 (IC50 = 27 nM) were also found to potentinhibitors against 5�-reductase type II [206].

eroids

dad(ti5smi


Kato and coworkers synthesized a series of pyrrole butyric aciderivatives and evaluated them for inhibitory activity on humannd rat steroid 5�-reductase. In case of para-aminobenzoyl pyrroleerivatives (353–356), the introduction of ethyl (354) or isopropyl355) at C-4 increased the activity [IC50 being 32 and 9.2, respec-ively], whereas the replacement with carboxyl (356) resultedn compounds with decreased inhibitory activity against human�-reductase enzyme, indicating steric restriction in the bindingite of the enzyme. Compound with m-amino benzyl pyrrole (357)oiety was found to be more active than the corresponding para

somer (355) [IC50 = 3.2 nM].

75 (2010) 109–153 145

Compound 358, having 2-hexyloctylamino group, was found tobe most potent inhibitor among the compounds with IC50 being0.60 nM against human and 5.8 nM against rat 5�-reductase [207].A novel series of indole-3-alkanoic acids with varied N-benzylsubstituents were also synthesized. Amongst these 4-[1-(6,6-dimethyl-6H-dibenzo [b,d] pyran-3-yl) methyl indol-3-yl]-butyricacid (359; FR119680) displayed very high inhibitory activity againstrat prostatic 5�-reductase (IC50 = 5.0 nM) [208].


dImm0

s5pwhinwr

Igarashi et al. found a novel series of phenoxybenzoic acidserivatives as potent inhibitors of human prostatic 5�-reductase.

t was found that introduction of a chloro (360), fluoro (361) orethoxy (362) group at 3-position of benzene ring (R1) leads to for-ation of compounds with high inhibitory activity with IC50 being

.87, 0.67 and 0.56 nM, respectively [209].

A series of indoline and aniline derivatives have also beenynthesized so as to inhibit both human and rat prostatic�-reductase. Among the indoline series, 3-chloro-4-{[1-(4-henoxybenzyl)indolin-5-yl]oxy}benzoic acid (363; YM-36117)as found to be the most potent inhibitor against human enzyme
aving an IC50 value of 5.3 and 46 nM against the rat enzyme while
n aniline series, 3-chloro-4-{4-[N-(4-phenoxybenzyl)amino] phe-oxy}benzoic acid (364) turned out to be most potent inhibitorith an IC50 against human and rat enzyme as 10 and 5.5 nM,

espectively [210].

75 (2010) 109–153

33. Bisubstrate inhibitors

Ishibashi et al. synthesized a series of novel benzofuran deriva-tives with both carboxy and 5- or 6-diphenylmethylcarbamoylgroups and their inhibitory activities against rat and human testos-terone 5�-reductase were tested in vitro. The derivatives were moreactive against human type I enzyme than against type II enzyme.The 6-carbamoyl derivative such as 365 tended to be more potentthan the 5-carbamoyl ones such as 366 with 365 being the mostpotent compound having IC50 value of 37.9, 50 and 340 nM againstrat, human type I and human type II isozymes [211].

Later, they also synthesized a series of 2-phenylbenzofuranderivatives with a carbamoyl, alkylamino, or alkyloxy group atthe 5 or 6 position of the benzofuran ring. It was found thatcarbamoyl derivatives had more potent inhibitory activities thanthe alkylamino or alkyloxy derivatives against the rat enzyme andthe 6-carbamoyl derivatives tended to be more potent than the5-carbamoyl ones. The 6-carbamoyl and 6-alkylamino derivativeswere found to be more potent inhibitors against human type Ienzyme than type II but on whole compounds were found not tobe selective [212]. The non-steroidal o-hydroxyaniline (261; ONO-3805) was a weak compound in vitro versus human 5�-reductaseII. It is a bi-substrate inhibitor in which the butanoic acid moietyis thought to be localized in the region of the phosphate group ofNADPH and the lipophilic part could be orientated in the regionof the steroidal C and D-ring, thus occupying the hydrophobicpocket 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 toprepare the derivatives of 261 and subsequently C-3 acylindole(367) was prepared which had improved potency versus bothhuman 5�-reductase enzymes. The benzodioxolane (368) adoptsa similar minimum conformation to the ether (367) and provedto be a potent dual inhibitor of both 5�-reductase enzymes.In common with the steroidal carboxylic acid inhibitors, these
compounds require the carboxylic acid moiety for potency andthe 3-acylindole motif was found to be crucial for dual activitypresumably by allowing access to both the conformations 369and 370. The corresponding 2-methyl analog 371 which adoptsconformation 370 due to the presence of methyl group on the


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

Compounds Rat 5�-reductase,IC50 (nM)

Human type I5�-reductase, IC50

(nM)

Human type II5�-reductase, IC50

(nM)

iImbr5avmb

In search of novel non-steroidal mimics of steroidal inhibitorsof 5�-reductase, 4-(2-phenylethyl)cyclohex-1-ene carboxylic acidswere synthesized with different substituents in para position ofthe phenyl ring such as N,N-diisopropylcarbamoyl (373), phenyl,

367 1 40 4368 9 25 23371 588 10 6300ONO-3805 (225) 1.7 – 256

ndole ring was found to be a selective inhibitor of 5�-reductase(Table 37) [215]. FK-143 (372) 4-[3-[3-[bis (4-isobutylphenyl)ethyl amino] benzoyl]-lH-indol-l-yl] butyric acid was disclosed

y Sawada et al. as a potent dual inhibitor of both human 5�-eductase isozymes. It inhibited in vitro human and rat prostatic�-reductase in a dose-dependent manner with an IC50 of 1.9nd 4.2 nM, respectively, in a non-competitive fashion while inivo showed potent inhibitory activity against castrated young ratodel. This compound can be a potential drug for the treatment of

enign prostatic hyperplasia [216–218].

75 (2010) 109–153 147

34. Miscellaneous non-steroidal inhibitors

1 eroids

ppo

hLta(5oer(a


henoxy, etc. They turned out to be good inhibitors of the humanrostatic 5�-reductase isozyme II with 373 being the most potentne (IC50 = 760 nM) [219].

Fan et al. evaluated a series of umbelliferone (7-ydroxycoumarin) derivatives as inhibitors of 5�-reductase I onNCaP cells. The coumarin skeleton was considered as a mimetic ofhe proposed transition state of the natural substrate and as wells bioisostere of quinolin-2-one 1′,1′-dimethylallyloxycoumarin374) showed potent inhibitory activity (IC50 = 1.3 �M) for the�-reductase I. This was possibly a result of conformational effectsf geminal dimethyl group. 8-Allyl-7-hydroxycoumarin (375) alsoxhibited potent inhibitory activity (IC50 = 0.99 �M) against 5�-eductase I enzyme. Introduction of a carbonyl group at 7-position376) resulted in only a slight increase in 5�-reductase I inhibitoryctivity (IC50 = 0.49 �M) [220].

75 (2010) 109–153

Due to the excellent estrogen receptor binding affinity of aseries of 2′,6′-disubstituted 4-hydroxy-4′-hydroxymethyl biphenylderivatives Lesuisse et al. designed various biphenyls as surrogatesof the steroidal backbone. They hypothesized that by introducingappropriate 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 being71 and 9.8 nM, respectively [221].

Chen et al. evaluated isoflavonoids as potential non-steroidalinhibitors of rat 5�-reductase by using the hypothetical phar-macophore of 5�-reductase. They proposed that although thesecompounds (379–382) were inhibitors of rat 5�-reductase in the

range of 27–49 �M they could be evaluated as human 5�-reductaseinhibitors [222].

eroids

IaM(w

3

crpwiapwiwhdpifbacnaasli

A

aI

R


Hasoda et al. in 2007 designed and synthesized novel type5�-reductase inhibitors by using 3,3-diphenylpentane skeletons a substitute for the usual steroid skeleton. 4-(3-(4-(N-ethylacetamido) phenyl) pentan-3-yl) phenyldibenzylcarbamate

383) was found to be a competitive 5�-reductase type I inhibitorith the IC50 value of 0.84 �M among the series [223].

5. Conclusion and future ahead

Finasteride (13) and Dutasteride (27) are the only two steroidallinically used drugs that have evolved from nearly 40 years ofesearch on steroids as 5�-reductase inhibitors but many com-ounds have shown promising results such as Epristeride (163)hich is in clinical trials. Combination therapy of 5�-reductase

nhibitors with various �-blockers like terazosin, alfuzosin and dox-zosin has been highly successful in the management of benignrostatic hyperplasia and combinations of 5�-reductase inhibitorsith anti-inflammatory agents have been tried successfully and it

s expected that this trend will continue. But the most challengingork will be the purification of 5�-reductase, for which all effortsave failed so far because of the unstable nature of the enzymeuring purification, leading to a loss of activity. Ligand-based com-arative pharmacophore development using the known potent

nhibitors could provide an insight into the structural requirementsor the possible inhibitors of 5�-reductase [224]. Data obtainedy such techniques could be used for developing more potentnd selective inhibitors that can be manufactured by pharma-eutical industries at a lower cost. Meanwhile, various classes ofon-steroidal inhibitors have also emerged. It would be encour-ging to see if any of these molecules such as FK-143 (372) will bevailable for clinical use. Meanwhile, synthesis of steroidal and non-teroidal derivatives will continue in search of a more potent andess toxic inhibitor of 5�-reductase. The basic research describedn this review article will assist in this process.

cknowledgements

Authors Saurabh Aggarwal and Abhilasha Verma gratefullycknowledge University Grants Commission (UGC, New Delhi,ndia) for providing fellowship to carry out research work.

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An overview on 5 -reductase inhibitors · b I. S. F College of Pharmacy, Ferozepur Road, Moga, Punjab 142001, India article info Article history: Received 13 July 2009 Received in