Structure-based design, synthesis and in vitro characterization of potent 17β-hydroxysteroid dehydrogenase type 1 inhibitors based on 2-substitutions of estrone and D-homo-estrone

  Structure-based design, synthesis and in vitro characterization of potent17 b -hydroxysteroid dehydrogenase type 1 inhibitors based on 2-substitutionsof estrone and D-homo-estrone Gabriele Möller a , Dominga Deluca a , Christian Gege b,  , Andrea Rosinus a , Dorota Kowalik a , Olaf Peters b,  ,Peter Droescher b,  , Walter Elger c,§ , Jerzy Adamski a, * , Alexander Hillisch c, – a Helmholtz Zentrum München, Institute of Experimental Genetics, Genome Analysis Center, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany b  Jenapharm GmbH & Co. KG, Medicinal Chemistry, Otto-Schott-Str. 15, 07745 Jena, Germany c EnTec GmbH, Adolf-Reichwein-Str. 20, 07745 Jena, Germany a r t i c l e i n f o  Article history: Received 22 July 2009Revised 27 September 2009Accepted 29 September 2009Available online 3 October 2009 Keywords: Steroid metabolismCancerInhibitorsMolecular dockingStructure-based drug design a b s t r a c t In search for specific drugs against steroid-dependent cancers we have developed a novel set of potentinhibitors of steroidogenic human 17 b -hydroxysteroid dehydrogenase type 1 (17 b -HSD 1). The X-raystructureof17 b -HSD1incomplexwithestradiolservedasbasisforthedesignoftheinhibitors.2-Substi-tutedestroneandD-homo-estronederivativesweresynthesizedandtestedfor17 b -HSD1inhibition. Thebest17 b -HSD1inhibitor, 2-phenethyl-D-homo-estrone, revealedanIC 50  of15nMinvitro. Theinhibitorypotency of compounds is comparable or better to that of previously described inhibitors. An interactionwithin the cofactor binding site is not necessary to obtain this high binding affinity for substancesdeveloped.   2009 Elsevier Ltd. All rights reserved. Human diseases can be treated by manipulation of selectivetargets contributing to pathogenesis. Several such targets are de-fined as enzymes including 17 b -hydroxysteroid dehydrogenases(17 b -HSDs). 1,2 The latter control the biological potency of steroidhormones by redox reactions at position 17 of the steroid scaf-fold. 3–5 The 17 b -HSDs belong to the short-chain dehydrogenase/reductase superfamily (SDR) 6 except for 17 b -HSD 5 which is analdo-ketoreductase. 7 Observations on the prognostic value of 17 b -HSDs in breastcancer, 8–10 prostate cancer, 11 and in endometriosis 12–14 haveboosted research on these enzymes. Approaches against breastand prostate cancers involve the development of new safe and17 b -HSD-specific drugs. 15–18 Several strategies include 17 b -HSD1 as a target and drug design has been facilitated by the knowncrystal structure of the enzyme. 19 An effective inhibitor of conver-sion of estrone to estradiol by 17 b -HSD 1 should deplete activehormone from the signal transduction pathway.Among naturally available substances the phytoestrogens arethe most potent inhibitors of 17 b -HSDs 20,21 but unfortunatelytheyare often non-specific. 22 Therefore, distinct highly specific 17 b -HSD 1 inhibitors have been developed. 14,23 Strategies for that in-cluded modifications of the steroid scaffold at positions 6, 16,and/or 17 16,24–28 and the substitution of hydroxyl moieties withsulfamates 29,30 orfluorine. 31 Evenhybridinhibitorsbasedonestra-diol derivatized with adenosine were reported. 17,32,33 Non-steroi-dal inhibitors of 17 b -HSD 1 have as well been identified. 34 However, none of the published inhibitors has progressed to clini-cal trials and further research is necessary.In this work we present the development of an inhibitor for17 b -HSD 1 for treatment of estradiol-dependent diseases as de-scribed in our patents. 35,36 We show the design, synthesis, andin vitro characterization of several 2-substituted derivatives of es-trone and D-homo-estrone. The main difference from previous re-portsisthesubstitutionofestroneandD-homo-estroneinposition2 at the aromatic A-ring. The X-ray structure of 17 b -HSD 1 showsan unoccupied lipophilic subpocket there and modifications at C2were only marginally pursued so far. 16,37 In addition, these 2-substituted inhibitors should show reduced intrinsic estrogenici- 0960-894X/$ - see front matter   2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.bmcl.2009.09.113 *  Corresponding author. Tel: +49 89 3187 3155; fax: +49 89 3187 3225. E-mail address:  adamski@helmholtz-muenchen.de (J. Adamski).  Present address: X2-Pharma GmbH,Im Neuenheimer Feld 584, 69120 Heidelberg,Germany.  Present address: Bayer Schering Pharma AG, Müllerstraße 178, 13353 Berlin,Germany. § Present address: Schorlemerallee 12B, 14195 Berlin, Dahlem, Germany. – Present address: Bayer Schering Pharma AG, Apratherweg 18a, 42096 Wuppertal,Germany. Bioorganic & Medicinal Chemistry Letters 19 (2009) 6740–6744 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl  ty, 37 since these do not fit well into the ER  a  binding site, 27 andshould posses altered properties with respect to phase II metabo-lism, due to shielding of the phenolic 3-hydroxy group. 27 Compounds  1  (estrone, E1) and  6  (Table 1) are commerciallyavailable while the synthesis of compounds  2 – 4 38 ,  5 37 ,  7 – 8 39 hasbeen published in the literature. The synthesis of the other deriva-tives was performed as described by us in full in our patents. 35,36 Inbrief,incorporationofthe2-pentanoylmoietyinto3-methoxyes-tra-1,3,5(10)-trien-17-one was accomplished under Friedel–Craftsconditions with valeroyl chloride and aluminium trichloride at5  Ctogivethe2-acylatedintermediate. 40 Cleavageofthe3-methyletherwithhydrobromideinaceticacidunderrefluxaffordedprod-uct  9 . Derivative  10  was synthesized from 3-acetoxy-2-iodo-estra-1,3,5(10)-trien-17-one 38 via a Sonogashira coupling with phenyl-acetyleneandsubsequentdeacetylation.Hydrogenationoftheacet-ylated 2-phenethynyl intermediate with palladium on charcoalfurnishedthe2-phenethyl-substitutedderivative 11  (Scheme1).Modification of the steroidal core towards the 14,15-dehydroderivative  12  was accessible by application of Saegusa’s generalprocedure to compound  6 . 41 The 14 a ,15 a -cyclopropa[ a ]estra-1,3,5(10),8-tetraene core 42 was substituted in the 2-position by ortho -lithiation similar as previously described 43 to give product 13 . Incorporation of the 6-oxo moiety towards compound  14  wasaccomplished with chromium trioxide in acetic acid as describedwith similar derivatives previously. 44 Halogenation of 18-homo-1,3,5(10)-trien-17-one was executedas described for derivative  2  to give compound  15 . 38 Fluorinationwas accomplished by silylation of ketone  2  and reaction of theresultingenoletherwith N  -fluoropyridiniumtriflate(NFPT)togivecompound  16  (Scheme 2). 45 Sodium methoxide catalyzed epimer-ization 45 of derivative  16  in methanol afforded an equimolar mix-ture, wherein the 16 b -fluoro derivative  17  was separated by HPLCin 33% yield.The D-homo moiety (Table 2) was incorporated via a modifiedTiffenau–Demjanow ring expansion, starting from the 17-ketoderivatives. 46,47 For example, reaction of   1  with trimethylsulfonium iodide andpotassium  tert  -butylate afforded the oxirane, which was openedwith sodium azide and then cyclized with trimethylsilyliodide toafford the D-homo derivative  18  in high overall yield (Scheme 3).Starting with compound  6 , product  22  was obtained on thesame route. After acetylation of compound  18 , incorporation of the halogen moiety in 2-position was accomplished via an  ortho -thallation as described previously, 38 albeit in lower yield (Scheme4). Subsequent deprotection furnished the derivatives  19 ,  20 , and 21 , respectively. The 2-allylated product  23  was obtained via theroute described for the corresponding estrone derivative (Scheme3). 39 Starting material  18  was reacted with allyl bromide and ce-sium carbonate as base in  N,N  -dimethylformamide to give the 3- O -allyl intermediate, which was rearranged in refluxing diethylan-iline to afford a mixture of the 2-allyl and 4-allyl isomer (ratio  1:2).SeparationbyHPLCfurnishedthedesired2-allylsubstitutedproduct  23 . Derivative  24  was synthesized similar as describedabove for derivative  11  (Scheme 4).  a -Fluorination of ketone  19 with Accufluor  NFTh was conducted as previously described 48 and furnished a diastereomeric mixture, which was separated byHPLC to give pure compounds  25  and  26 .Inhibition of catalytic activity of human 17 b -HSD 1 towards E1was assessed as srcinally described by us and a detailed descrip-tion for that is given in the Supplementary data section. 31,49 TheX-raystructureof17 b -HSD1incomplexwithestradiol(E2)and NADP + (PDB entry code 1FDT) 19 was used for the docking  Table 1 Inhibition of 17 b -HSD 1 by 2-substituted estrone derivatives HHHR 2 HOOR 16 R 18 OOHO 12 OHHOHO 13 14 OHHHOHOO Compd R  2 R  16 R  18 % Inhibition @ 2 l M IC 50  (nM) 1  (E1) H H H 90 109 2  Cl H H 95 140 3  Br H H 95 233 4  NC H H 96 148 5  Et H H 86 89 6  MeO H H 70 207 7  Allyl H H 94 109 8  n -Propyl H H 91 545 9  CH 3 (CH 2 ) 3 CO H H 85 218 10  Ph-C „  C H H 90 56 11  Phenethyl H H 97 47 12  — — — 84 633 13  — — — 97 193 14  — — — 80 392 15  Cl H Me 86 121 16  Cl  a -F H 99 101 17  Cl  b -F H 92 35 11 HHH AcOHHH AcOaHHHHOb, cOIOO Scheme 1.  Reagents and conditions: (a) PhC „  CH, Pd(OAc) 2 , PPh 3 , CuI, NEt 3 /THF(3:2), rt, 89%; (b) H 2  (1bar), Pd/C, EtOAc, rt, 3h; (c) NaOMe, MeOH/THF, rt, 89% forboth steps. 17 HHHHOHHHHOa, b ClHHHHOClcOClOO 162 FF Scheme 2.  Reagents and conditions: (a) TMSOTf, NEt 3 , toluene, reflux; (b) NFPT,CH 2 Cl 2 , rt, 1N HCl; (c) NaOMe, MeOH, rt, 11d, 33%. G. Möller et al./Bioorg. Med. Chem. Lett. 19 (2009) 6740–6744  6741  experiments to support structure–activity relationships (SAR)studies and design of inhibitors. The 3D-structures of the steroidalcompoundsweregeneratedwithin SYBYL  6.7(TriposInc. SYBYL  , 2000)and energy minimized using the MMFF94 force field. 50 The  FLEXX program version 1.8 51 interfaced with  SYBYL   was used to performthe docking experiments to 17 b -HSD 1. Glu282 was treated ascharged residue, whereas His221 was kept uncharged. All default FLEXX  parameters, as implemented in the 6.7 release of   SYBYL  , wereused. Substrate binding was analyzed using the program  MOLCAD in  SYBYL  .Thecrystal structureof 17 b -HSD1incomplexwithE2was pre-viously applied in molecular dynamics simulations and ligand–protein docking studies of non-steroidal 52–54 as well as steroidalderivatives. 16,28,30,55 In the latter studies mainly the region in the16and17positionofthesteroidwasinvestigated,whichpointsto-wardstheNADP + cofactor.Inourstudy,wefocusedonposition2of estrone and D-homo-estrone. From the surface of the substrate-binding pocket in the 17 b -HSD 1 X-ray structures it is clearly vis-ible that the position 2 of E2 is located in a lipophilic environmentconsisting of the side-chains of Val143, Met147, Phe259, Leu262and Met279. In this region the substrate binding pocket forms alipophilic tunnel to the exterior of the protein. The existence of such a lipophilic tunnel inspired us to synthesize steroidal com-pounds substituted with lipophilic residues (such as halogen or al-kyl) in position 2. In addition, fluorine substituents wereintroducedinposition16 a and b  tostabilizethe17carbonylgroupof estrone from metabolic attack. The five-membered D-ring wasalso enlarged to a six-membered (D-homo) ring.Several 2-substituted estrone derivatives were tested for theirability to inhibit recombinant 17 b -HSD 1 activity (Table 1). Struc-ture–activity relationships focused mainly on the role (size andpolarity) of substitutions at position 2 of the steroidal A-ring andsome modifications of the B- and D-ring (Fig. 1).Based on the observed IC 50 -values all compounds in this seriesare submicromolar to nanomolar inhibitors of 17 b -HSD 1. To facil-itatenormalizedcomparisonweanalyzedthenatural substratees-trone (E1) which shows under the same conditions an IC 50  of 109nM. Compared to halogen/pseudohalogen substituents suchas chlorine, bromine and carbonitrile (compounds  2 – 4 ) in 2-posi-tion do not lead to increased inhibition. The same is true for the18-homo estrone derivative  15  and the 2-methoxy-derivatives(compounds  6 ,  12 – 14 ). We had made similar observations for17-fluorine-substituted estrogens in a previous study. 31 It is pro-posedthatanintramolecularhydrogenbondbetweenthe3-hydro-xylgroupandtheoxygenatomofthe2-methoxysubstituentofthesteroid reduces binding to His221 and Glu282 of 17 b -HSD 1 andpositions the methoxy group in the plane of the steroidal A-ring.This might lead to repulsive interactions with Phe259. This intra-molecular hydrogen bond and the respective conformation of themethoxy group is observed in a number of small molecule X-raystructures bearing the  O -methoxy-phenyl ring system. 56 Modification of compound  6  with an additional carbonyl groupin position 6 (compound  14 ) or double bond in position 14–15(compound  12 ) leads to diminished inhibition while introductionof a 8–9 double bond together with a 14 a ,15 a -cyclopropyl substi-tuent does not alter the IC 50  in comparison with compound  6 . Thedocking studies suggest that the 6-oxo group in compound  14 could lead to repulsive interactions with Ser222 and Tyr218.Slightly increased inhibition was observed with the 2-ethyl sub-stituent (compound  5  with IC 50  =89nM) while 2-allyl, propyl,and pentanoyl resulted in equally potent or weaker inhibitors.Especially the terminal CH 3 -group of the 2-ethyl substituent isproposed to fill a subpocket formed by Val143, Met147, Leu149,Phe259, and Leu262 of 17 b -HSD 1 while the other substituentsare longer and interact with Leu262, which is more exposed tothe solvent. A significant increase in inhibitory activity comparedto E1 was observed for the 2-phenethynyl and 2-phenethyl sub-stituents (compounds  10 , IC 50  =56nM and  11 , IC 50  =47nM).These longer substituents are proposed to form lipophilic contactswith Leu262 and Phe259, which might explain the increasedinhibition.The most potent inhibitor in this series was obtained by anadditional substitution of compound  2  (2-chloro-estrone) in posi-  Table 2 Inhibition of 17 b -HSD 1 by 2-substituted D-homo-derivatives of estrone HHHR 2 OR 17 HO Compd R  2 R  17 % Inhibition @ 2 l M IC 50  (nM) 18  H H 91 30 19  Cl H 100 77 20  Br H 100 73 21  I H 100 123 22  MeO H 92 85 23  Allyl H 97 32 24  Phenethyl H 95 15 25  Cl  a -F 86 87 26  Cl  b -F 100 126 HHHHOO 18 HHHHOOd, ea, b, cHHHHOO 123 Scheme 3.  Reagents and conditions: (a) MeSI (2equiv), KO t  Bu (2.2equiv), DMSO,rt, 80%; (b) NaN 3  (1.3equiv), DMF, 60  C, 57%; (c) NaI, Me 3 SiCl, CH 3 CN, rt, 75%; (d)AllBr, Cs 2 CO 3 , DMF, 60  C, 3h, 89%; (e) PhNEt 2 , reflux, 8h, 34% (and 4-allyl isomer). HHHHOO 18 HHH AcOOa, b IHHHHOOc, d 24 Scheme 4.  Reagents and conditions: (a) Ac 2 O, pyridine, cat. DMAP, rt, quant.; (b)Tl(OCOCF 3 ) 3 , TFA, 10  C, then KI, 40%; (c) PhC „  CH, Pd(OAc) 2 , PPh 3 , CuI, NEt 3 /THF(3:1), rt, 80%; (d) H 2  (1bar), Pd/C, EtOAc/THF, rt; NaOMe, MeOH/CH 2 Cl 2 , rt, 65%.6742  G. Möller et al./Bioorg. Med. Chem. Lett. 19 (2009) 6740–6744  tion 16 with fluorine. 2-chloro-16 b -fluorine-estrone (compound 17 )issignificantlymorepotent(IC 50  =35nM)thanthe16 a -isomer(IC 50  =101nM). An electrostatic repulsive interaction between thepartially negatively charged 16 a -fluorine substituent and theamide carbonyl oxygen of NADP + (distance   3Å) might explainthis difference of the two stereoisomers.The structure–activity study was extended to compounds withanother steroid-like scaffold, the D-homo-estrone (Table 2), inwhich the five-membered D-ring of estrone is expanded to an ali-phatic 6-ring. This modification was purely driven by the favour-able synthetic accessibility of these compounds. It has beenshown that androstanedione, androstenedione, and testosteronebind to 17 b -HSD 1 in a reverse binding mode in comparison toE2. 57,58 When bound substrate conformations are compared, thesix-membered A ring of the androgens overlays approximatelywith the five-membered D-ring of E2. This observation allows forthe assumption that D-homo-estrogens probably behave likeandrogens on binding to 17 b -HSD 1. The lead compound D-homo-estrone  18 , having a six-membered A and D-ring (Table 2),revealed an IC 50  of 30nM which is even better than that obtainedwith the most potent compound  17  from the series based on 2-substituted E1. This is surprising, because the SAR around the 2-position between the two lead series (estrone and D-homo-es-trone) is completely parallel (Tables 1 and 2). Chlorine, bromine,and methoxy modifications lead to weaker inhibitors. Allyl is neu-tral in comparison to compound  18 , whereas 2-phenethyl in-creases inhibition and represents the most potent compound inthis study (compound  24 , IC 50  =15nM). This suggests a very sim-ilar binding mode of the two series which is also supported by ourdockingexperiments. In contrast to the estrone series (compounds 16  and  17 ), fluorine substitution in the position vicinal to the car-bonylmoiety(position17)leadstoweakerinhibitorsthanplainD-homo-estrone 18 . Thiseffectcannotbeexplainedindetailwiththeapplied docking procedures. Figure 1.  Molecular modelling. Lipophilic surface areas are colored brownish, green corresponds to neutral and blue to hydrophilic parts of the protein surface. Leu149 andPhe259 are not depicted since they are positioned far below and above the steroid plane. (Top) Substrate binding pocket of 17 b -HSD 1 in complex with estradiol and NADP + according to PDB data 1FDT. (Bottom) Modelled complex of 2-phenethyl-D-homo-estrone  24  with 17 b -HSD 1 and NADP + . G. Möller et al./Bioorg. Med. Chem. Lett. 19 (2009) 6740–6744  6743  Binding affinity of human hER  a  (hER  a ) was tested for selected2-substitutedestrogenderivativesandcomparedtothatofestroneandestradiol (Table3). 59 The bindingaffinityandaccordinglyestr-ogenicity is reduced by 100- to 1000-fold making the substancesapplicable inhibitor candidates as expected. 27,37 In conclusion, novel and potent inhibitors of 17 b -HSD 1 wereidentified applying structure based design, chemical synthesisand biochemical testing of the recombinant enzyme in an iterativemanner.Theinhibitorswerederivedfromestrone,thenaturalsub-strate of 17 b -HSD 1. The X-ray structure of 17 b -HSD 1 guided thedesignof thecompounds. Themost potentcompoundischaracter-ized by an IC 50  in the low nanomolar range, sevenfold better thanthat of estrone. Withrespecttoinhibitorypotency, thecompoundsare comparable to previously describedinhibitors such as non-ste-roidal compounds or compounds that are linked to cofactors inpositions16and17. 16,26,34e However,theincreaseinpotencyisob-tained by only adding relatively little molecular weight. Generally,thestrategypresentedinthisworkmightbebettersuitedtoobtainmore drug-like molecules for oral administration.  Acknowledgment This project was supported by BMBF-Project ‘Bioinstrumente Jena 0312255’. Supplementary data Supplementary data associated withthis article can be foundinthe online version at doi:10.1016/j.bmcl.2009.09.113. References and notes 1. Hardy, L. W.; Peet, N. P.  Drug Discovery Today  2004 ,  9 , 117.2. Hopkins, A. L.; Groom, C. R.  Nat. Rev. Drug Disc.  2002 ,  1 , 727.3. Möller, G.; Adamski, J.  Mol. Cell. Endocrinol.  2009 ,  301 , 7.4. Vihko,P.;Härkönen,P.;Soronen, P.;Törn,S.;Herrala,A.;Kurkela, R.;Pulkka, A.;Oduwole, O.; Isomaa, V.  Mol. Cell. 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