Androsterone 3 -Substituted Derivatives as Inhibitors of
Type 317 -Hydroxysteroid Dehydrogena
Be
ombrelleatrice Tche dam Ngatcha,a Van Luu-The b and Donald Poirier a,*a
Medicinal Chemistry Division,Oncology and Molecular Endocrinology Rearch Center,
Laval University Medical Center (CHUL)and Laval University,2705Laurier Blvd.,Que
Âbec,Canada G1V 4G2b
囫囵MRC Group in Molecular Endocrinology,Oncology and Molecular Endocrinology Rearch Center,
Laval University Medical Center (CHUL)and Laval University,2705Laurier Blvd.,Que
万圣节快乐英文
Âbec,Canada G1V 4G2Received 8June 2000;accepted 5September 2000
AbstractÐAndrosterone derivatives substituted at position 3were synthesized starting from dihydrotestosterone in a short
quence of reactions.They proved to be potent inhibitors (IC 50=57±147nM)of type 317b -hydroxysteroid dehydrogena,a key enzyme of steroidogenesis,which catalyzes the transformation of androstenedione to steroid active androgen testosterone.#2000Elvier Science Ltd.All rights rerved.
The type 317b -hydroxysteroid dehydrogena (type 317b -HSD),also called testicular 17b -HSD or andro-genic 17b -HSD,is found principally in the microsomal fraction of the testis.1With NADPH as a cofactor,it catalyzes the reduction of 4-androstene-3,17-dione (Á4-dione)to testosterone (T),2À4which is further converted to dihydrotestosterone (DHT)by 5a -reducta (Scheme 1).5Thus,type 317b -HSD plays an important role in the formation of active androgens (T and DHT),which are both involved in the pathophysiology of various andro-gen-nsitive dias,such as benign prostatic hyperplasia,prostate cancer,6acne,7hirsutism,8and male-pattern baldness.9
During our e orts to develop inhibitors of type 117b -HSD,10À12and type 217b -HSD,13À15we became inter-ested in the development of type 317b -HSD inhibitors.In his evaluation of the ability of 20steroidal com-pounds to inhibit the type 317b -HSD activity in microsomal preparations of canine testis,Pittaway sug-gested the requirement of a 17-keto group and a ster-oidal unaromatized A-ring to inhibit the enzyme.16More recently,a screening study with 80steroids of di erent class led us to
camus
consider the C19steroid androsterone (ADT)as a potential starting nucleus to develop inhibitors of type 317b -HSD.17Bad on our results obtained during the development of inhibitors of type 117b -HSD,11,12ADT derivatives substituted at position 16were synthesized,but they turned out to be only weak inhibitors of type 317b -HSD.18We then decided to experiment with position 3of the ADT nucleus to develop new inhibitors.We herein report the chemical synthesis of 3b -substituted ADT derivatives and their ability to inhibit the type 317b -HSD activity.
0960-894X/00/$-e front matter #2000Elvier Science Ltd.All rights rerved.P I I:S 0960-894X(00)00517-
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Bioorganic &Medicinal Chemistry Letters 10(2000)
2533±2536
Scheme 1.Enzymatic steps involved in biosynthesis of testosterone (T)and dihydrotestosterone (DHT)from 4-androstene-3,17-dione (Á4-dione).*Corresponding author.Tel.:+1-418-654-2296;fax:+1-418-654-2761;e-mail:donald.poirier@crchul.ulaval.ca
Chemistry
DHT was ud as starting material for the synthesis of the target compounds5±15depicted in Scheme2.The 17b-hydroxy group of DHT was®rst protected as a tert-butyldimethylsilyl ether,using TBDMS-Cl and imida-zole in DMF to a ord compound2in a91%yield.The C3-carbonyl group of17b-T
BDMS-DHT(2)was then subjected to various alkylating reagents(Grignard or lithium reagents)to give a mixture of3and4.In most of the cas(methyl,n-propyl,n-hexyl,n-octyl,cyclo-hexyl,phenyl,and phenylmethyl derivatives),a com-mercially available Grignard reagent was ud and the reaction was performed at0 C in dry THF.For cyclo-hexylmethyl and phenylethyl derivatives,the Grignard reagent was generated in situ,by a well known proce-dure described by Smith,19using a magnesium amalgam and the corresponding halide.For cyclohexylethyl deri-vative,the lithium reagent was generated in situ with t-BuLi and cyclohexylethyl bromide,in a mixture of die-thylether:pentane(2:3),according to the procedure described by Bailey and Puzalan.20Finally,a commer-cially available lithium reagent was ud for the synth-esis of s-butyl derivative.Generally,a mixture of the two stereoisomers at position3was obtained,the pro-portions varying according to the nature of the alkyl group.21The two stereoisomers could be well di er-entiated on TLC,the3b-substituted derivative always being the less polar one.Hence,a paration by¯ash chromatography was done,that parated the3a-sub-stituted stereoisomer from the3b-substituted one and the quence of reactions was continued with the latter. Less than25%of starting material was also recovered in most cas.The TBDMS protective group of general compound3was then hydrolyzed with a2%HCl methanolic solution at room temperature.The resulting 17b-alcohol was directly oxidized by Jones'reagent to
a ord the desired ADT3b-substituted derivatives5±
15.22
2011年高考英语
Inhibition of Type317 -HSD
The inhibitory properties toward type317b-HSD activity of target compounds5±15were evaluated in transfected HEK-293cells by measuring the amount of labeled T formed from the natural labeled substrateÁ4-dione in the prence of NADPH as cofactor.The enzymatic assay was performed as described23and the results are summarized in Table1.The concentration of compound that produced50%of inhibition(IC50value) was determined from the inhibition curves.The ADT 3b-alkylated derivatives5±15were good inhibitors of type317b-HSD;they all showed an inhibitory activity higher than that ofÁ4-dione,when this natural sub-strate of the enzyme was ud as inhibitor itlf.The compounds5±15were also stronger inhibitors than ADT,which had been determined as the strongest type 317b-HSD inhibitor from our screening study.17In the n-alkyl ries,the best inhibitory activity was obtained for n-propyl derivative6.However,this activity fell as the longer3b side chain was made longer(IC50of 100nM and147nM for8and9,respectively).This suggested a length limit for this kind of substituent.This led us to synthesize and test branched substituents,such as the s-butyl d
erivative7.With an IC50value of73nM, compound7was as good an inhibitor as the other ana-logues in the n-alkyl ries(6and8).To test a sub-stituent with a well de®ned shape,we also evaluated cyclic derivatives.Cyclohexyl-ADT(10)and phenyl-ADT (13)gave almost the same inhibitory activity,with IC50 values of97and81nM,respectively.Adding a
methylene
Scheme2.Synthesis of ADT3b-substituted derivatives5±15.Reagents:(a)TBDMS-Cl,imidazole DMF,rt;(b)i.RMgBr(Cl)or RLi,THF,0 C.ii. Flash chromatography;(c)i.MeOH±HCl(2%),rt.ii.Jones'reagent(2.7M),acetone,0 C.
2534 B.TcheÂdam Ngatcha et al./Bioorg.Med.Chem.Lett.10(2000)2533±2536
group resulted in a gain of inhibitory activity,which was more important in the ca of phenyl:IC 50value of 87nM for cyclohexylmethyl-ADT (11)and 57nM for phe-nylmethyl-ADT (14).The addition of two methylene groups led to another gain of inhibitory activity in cyclohexyl ries (IC 50value of 60nM for cyclohex-ylethyl-ADT (12)),but led to a loss of inhibitory activity in the phenyl ries (IC 50value of 99nM for pheny-lethyl-ADT (15)).With an IC 50value of 57nM,3b -phenylmethyl-ADT (14)was the most potent type 317b -HSD inhibitor obtained in this study,it was 6-fold more powerful than ADT and 13-fold more powerful than Á4-dione,the natural substrate of the enzyme.In an attempt to correlate hydrophobicity and inhibi-tory activity,the LogP values were calculated for all compounds.24This logarithm of partition coe cient between n -octanol and water express the relative hydrophobicity of a compound.Á4-Dione and ADT,which gave the lowest inhibitory activities
of tested compounds,were less hydrophobic (LogP=3.5and 4.2,respectively)than inhibitors 5±15(LogP ranging from 4.4to 7.4),suggesting that hydrophobicity is required.On the other hand,compound 9,which happened to be the most hydrophobic with a LogP value of 7.4,showed only moderate inhibitory activity (IC 50=147nM).Thus,the prence of a hydrophobic substituent in position 3of ADT is important for good inhibition of type 317b -HSD,but a limitation was also obrved for this hydrophobicity thus implicating important steric e ects.
In conclusion,ADT 3b -substituted derivatives 5±15were synthesized and found to inhibit the steroidogenic
enzyme,type 317b -HSD.Interestingly,no inhibition of other reductive 17b -HSDs (type 1and type 5)was obrved at a 0.3m M concentration of compounds 5±15suggesting a speci®c inhibition.To the best of our knowledge,the ADT 3b -substituted derivatives con-stitute the ®rst inhibitors of type 317b -HSD ever syn-thesized.Further experiments are being carried out to optimize the inhibitory activity of the compounds and to determine the exact mechanism of inhibition.The results will be published later in a full paper with a complete description of the experimental procedure (chemical synthesis and enzymatic test).
Acknowledgements
We thank the Medical Rearch Council of Canada
etc(MRC)and Le Fonds de la Recherche en Sante
du Que
bec (FRSQ)for operating grants and fellowships.We are grateful to the Laboratory of Molecular Endo-crinology (Dr.F.Labrie,Director)for providing che-mical and biological facilities.We also thank Guy Reimnitz and Mei Wang for the enzymatic assay.
References and Notes
1.Andersson,S.;Geissler,W.M.;Patel,S.;Wu,L.J.Steroid Biochem.Mol.Biol.1995,53,37.lsp是什么意思
2.Peltoketo,H.;Luu-The,V.;Simard,J.;Adamski,J.J.Mol.Endocrinol.1999,23,1.
3.Penning,T.M.Endocrine-Related Cancer 1996,3,41.
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5.Li,X.;Calin,C.;Singh,S.M.;Labrie,F.Steroids 1995,60,430.
6.Labrie,F.;Dupont,A.;Be
langer,A.In Important Advances in Oncology ;DeVita,V.T.,Jr,Hellman,S.,Ronberg,S.A.,Eds.;Lippincott:Philadelphia,1985;pp 193±217.
7.Sansone,G.L.;Reisner,R.M.J.Invest.Dermatol.1971,56,366.
8.Kuttenn,F.;Mowszowicz,I.;Shaison,G.;Mauvais-Jarvis,P.J.Endocrinol.1977,75,83.
9.Bingham,K.D.;Shaw,D.A.J.Endocrinol.1973,57,111.10.Poirier,D.;Dionne,P.;Auger,S.J.Steroid Biochem.Mol.Biol.1998,64,83.
11.Tremblay,M.R.;Poirier,D.J.Steroid Biochem.Mol.Biol.1998,66,179.
12.Sam,K.M.;Boivin,R.P.;Tremblay,M.R.;Auger,S.;Poirier,D.Drug Des.Dis.1998,15,157.
13.Sam,K.M.;Auger,S.;Luu-The,V.;Poirier,D.J.Med.Chem.1995,38,4518.
14.Tremblay,M.R.;Luu-The,V.;Leblanc,G.;Noe l,P.;Breton,E.;Labrie,F.;Poirier,D.Bioorg.Med.Chem.1999,7,1013.
15.Sam,K.M.;Labrie,F.;Poirier,D.Eur.J.Med.Chem.2000,35,217.
16.Pittaway,D.E.Contraception 1983,27,431.河北省公务员考试历年真题
17.Poirier,D.;Labrie,F.;Luu-The,V.Me
Âdecine-Sciences (Suppl.2)1995,11,24.
18.Tche
dam Ngatcha, B.,PhD thesis,University Laval,Que
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Table 1.Inhibition of type 317b -HSD by ADT 3b -substituted deri-vatives 5±
15
Inhibition of type 317b -HSD (%)a
darkCompounds R
0.3m M 3m M IC 50(nM)Á4-Dione 3-Keto-4-ene
2478758Æ139ADT H 5088330Æ605CH 3
7293nd b 6CH 3(CH 2)2
899467Æ67CH 3CH 2(CH 3)CH
859073Æ58CH 3(CH 2)59396100Æ109CH 3(CH 2)78892147Æ2910Cyclohexyl 889397Æ311Cyclohexyl-CH 2939587Æ1912Cyclohexyl-CH 2CH 2
929360Æ1613Ph 889581Æ614PhCH 2909457Æ515
Ph CH 2CH 2
93
93
99Æ1
a Error Æ10%.b
Not determined.
B.Tche神盾局特工第三季13集
Âdam Ngatcha et al./Bioorg.Med.Chem.Lett.10(2000)2533±25362535
20.Bailey,W.F.;Punzalan,E.R.J.Org.Chem.1990,55,5404.
21.Tche dam Ngatcha,B.;Poirier,D.Synth.Commun.1999, 29,1065.
22.All compounds were characterized by1H NMR,13C NMR,FT-IR,MS,and elemental analysis.
23.Enzymatic assay(brie¯y):An expression vector encoding for type317b-HSD was transfected into human embryonal kidney(HEK)-293cells using the calcium phosphate proce-dure.25Cells were then sonicated in50mM sodium phosphate bu er(pH7.4),containing20%glycerol and1mM EDTA, and centrifuged at10,000g for1h to remove the mitochon-dria,plasma membranes,and cells fragments.Th
e supernatant was further centrifuged at100,000g to parate the micro-somal fraction and this was ud for measurement of type3 17b-HSD activities.The test was carried out at37 C for1h in 1mL of above reported bu er,containing2mM of cofactor (NADPH)and0.1m M of14C-Á4-dione(New England Nuclear,Boston,MA)and indicated concentration of inhibitors.The reaction was stopped by adding2mL of diethylether contain-ing10mM of unlabeledÁ4-dione and T.The metabolites were extracted with diethylether before being applied on silica gel 60TLC plates.TLCs were developed in a mixture of toluene and acetone(4:1).Substrates and metabolites were identi®ed by comparison with reference steroids,revealed by auto-radiography,and quanti®ed using the phosphorImager (Molecular Dynamics,Sunny Vale,CA).The percentage of inhibition and IC50values were then calculated.
24.The logarithm of partition coe cient of n-octanol/water (LogP values)were calculated by CS ChemDraw Pro(Cam-bridgeSoft Corporation,Cambridge,MA)using the Crippen's fragmentation method(Gho,A.K.;Crippen,G.M.J. Chem.Inf.Comput.Sci.1987,27,35).
25.Luu-The,V.;Zhang,Y.;Poirier,D.;Labrie,F.J.Steroid Biochem.Mol.Biol.1995,55,581.
2536 B.TcheÂdam Ngatcha et al./Bioorg.Med.Chem.Lett.10(2000)2533±2536
