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References Cited Deng et al. , Characterization of a selective inhibitor of the Parkin ( 56 ) son ' s disease kinase LRRK2. Nat Chem Biol . Apr. 2011 ; 7 ( 4 ) : 203 - 5 . OTHER PUBLICATIONS Deng et al. , Discovery of a benzo [ e ]pyrimido - [ 5 , 4 -b ] [ 1 , 4 ] diazepin 6 ( 11H )- one as a Potent and Selective Inhibitor of Big MAP Kinase [No Author Listed ], PubChem SID 235048169 . Feb. 13 , 2015. 1 . ACS Med Chem Lett . Mar . 10 , 2011; 2 ( 3 ) : 195 - 200 . Deng et al. , Structural determinants for ERK5 (MAPK7 ) and Retrieved on Oct . 24 , 2016 . Available at https: // pubchem .ncbi . nlm . leucine rich repeat kinase 2 activities of benzo [ e ]pyrimido - [ 5 , 4 nih . gov / substance /235048169 . b ]diazepine - 6 ( 11H ) -ones . Eur J Med Chem . 2013 ; 70 : 758 -67 . [ No Author Listed ] , PubChem SID 235671906 . Feb . 13 , 2015 . Elkins et al. , X -ray crystal structure of ERK5 (MAPK7 ) in complex Retrieved on Oct . 24 , 2016 . Available at https :/ /pubchem .ncbi . nlm . with a specific inhibitor . J Med Chem . Jun . 13 , 2013 ; 56 ( 11 ) : 4413 nih . gov / substance / 235671906 # section = Top > . 21. Anders et al. , Genome -wide localization of small molecules . Nat Filippakopoulos et al. , Targeting bromodomains : epigenetic readers Biotechnol . Jan . 2014 ; 32 ( 1 ) : 92 -6 . doi: 10 . 1038 /nbt . 2776 . Epub of lysine acetylation . Nat Rev Drug Discov. May 2014 ; 13 ( 5 ) :337 Dec . 15 2013 56 . doi: 10 . 1038 /nrd4286 . Epub Apr. 22 , 2014 . Bartholomeeusen et al. , Bromodomain and extra - terminal (BET ) Kavanagh et al. , the development of CNS - active LRRK2 inhibitors bromodomain inhibition activate transcription via transient release using property - directed optimisation .Bioorg Med Chem Lett. Jul. 1 , of positive transcription elongation factor b (P - TEFb ) from 7SK 2013 ; 23 ( 13 ) : 3690 -6 . small nuclear ribonucleoprotein . J Biol Chem . Oct. 19 , Krueger et al ., The mechanism of release of P - TEFb and HEXIMI 2012 ;287 (43 ) : 36609 - 16 . doi: 10 .1074 / jbc .M112 .410746 . Epub Sep . from the 7SK snRNP by viral and cellular activators includes a 5 , 2012 . conformational change in 7SK . PLoS One. Aug. 23 , Baud et al. , Chemical biology. A bump- and -hole approach to 2010 ; 5 ( 8 ) :e12335 . doi: 10 . 1371 / journal .pone .0012335 . engineer controlled selectivity of Bet bromodomain chemical Lotti et al. , Ultrasound of the male genital tract in relation to male probes . Science . Oct. 31, 2014 ; 346 (6209 ) :638 -41 . doi: 10 . 1126 / reproductive health . Hum Reprod Update . Jan . -Feb . 2015 ;21 ( 1 ) :56 science . 1249830 . Epub Oct . 16 , 2014 . 83 . doi: 10 . 1093 /humupd /dmu042 . Epub Jul . 19 , 2014 . Berge et al . , Pharmaceutical salts . J Pharm Sci. Jan . 1977 ;66 ( 1 ) : 1 Mckeown et al. , Biased multicomponent reactions to develop novel 19 . bromodomain inhibitors. J Med Chem . Nov. 13 , 2014 ;57 ( 21 ) : 9019 CAPLUS Database Result for Deng et al. , Structural determinants 27 . doi: 10 . 1021/ jm501120z . Epub Oct . 31 , 2014 . for ERK5 (MAPK7 ) and leucine rich repeat kinase 2 activities of Roberts et al. , A Bead - Based Proximity Assay for BRD4 Ligand benzo [ e ]pyrimido - [ 5 , 4 - b ]diazepine -6 ( 11H ) -ones . Eur J Med Chem . Discovery. Curr Protoc Chem Biol. Dec . 2 , 2015 ; 7 ( 4 ) : 263 - 78 . doi: 2013 ; 70 :758 - 67 . doi: 10 . 1016 / j .ejmech .2013 . 10 .052 . Epub Oct . 29 , 10 . 1002 /9780470559277 . ch150024 . 2013 . Accession No . 2013 : 1979798 . Abstract Only . Schroder et al. , Two -pronged binding with bromodomain -contain Chaidos et al. , Potent antimyeloma activity of the novel ing protein 4 liberates positive transcription elongation factor b bromodomain inhibitors I -BET151 and I -BET762 . Blood . Jan . 30 , from inactive ribonucleoprotein complexes . J Biol Chem . Jan . 6 , 2014 ; 123 ( 5 ) :697 -705 . doi: 10 . 1182 /blood -2013 -01 -478420 . Epub 2012 ;287 (2 ): 1090 - 9. doi : 10 . 1074 /jbc .M111 . 282855 . Epub Nov. 14 , Dec . 13 , 2013 . 2011. Cheng et al. , Adjudin disrupts spermatogenesis via the action of Smith et al. , The Bromodomain : A New Target in Emerging someunlikely partners : Eps8, Arp2/ 3 complex , drebrin E , PAR6 and Epigenetic Medicine . ACS Chem Biol. Mar. 18 , 2016 ; 11 ( 3 ) :598 14 - 3 - 3 . Spermatogenesis . Oct . 2011 ; 1 ( 4 ) : 291- 297 . Epub Oct. 1 , 608 . doi : 10 . 1021/ acschembio .5b00831 . Epub Dec . 3, 2015 . 2011 . Tanaka et al. , Inhibitors of emerging epigenetic targets for cancer Choi et al. , Brain Penetrant LRRK2 Inhibitor. ACS Med Chem Lett . therapy : a patent review (2010 - 2014 ) . Pharm Pat Anal. Aug. 9, 2012; 3 (8 ): 658 -662 . 2015 ; 4 ( 4 ) :261 - 84 . doi: 10 .4155 / ppa . 15 . 16 . Dawson et al. , Inhibition of BET recruitment to chromatin as an Wehner et al. , Effects of natalizumab , an alpha4 integrin inhibitor, effective treatment for MLL - fusion leukaemia . Nature. Oct . 2 , on fertility in male and female guinea pigs . Birth Defects Res B Dev 2011; 478 ( 7370 ) : 529 - 33 . doi: 10 . 1038 / nature10509 . Reprod Toxicol. Apr. 2009 ; 86 ( 2 ) : 108 - 16 . doi: 10 . 1002 /bdrb . 20191 . Delbroek et al. , Development of an enzyme- linked immunosorbent Zuercher et al. , Identification and structure -activity relationship of assay for detection of cellular and in vivo LRRK2 S935 phenolic acyl as selective agonists for the estrogen phosphorylation . J Pharm Biomed Anal. Mar. 25, 2013 ; 76 :49 -58 . related orphan nuclear receptors ERRbeta and ERRgamma. . J Med Delmore et al. , BET bromodomain inhibition as a therapeutic Chem . May 5 , 2005 ; 48 ( 9 ) : 3107 - 9 . strategy to target c -Myc . Cell . 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Provisional Application No. 61 / 334 , 991 , filed pounds and compositions for effecting male contraception . May 14 , 2010 ; 61 / 370 ,745 , filed on Aug . 4 , 2010 ; 61/ 375 , The invention also provides methods for using such com 863, filed on Aug . 22 , 2010 ; 61/ 467, 376 , filed on Mar. 24 , pounds and compositions in a male subject . 2011 ; and 61 / 467 ,299 , filed Mar. 24 , 2011. The contents of In one aspect , the invention provides methods for reduc these applications are hereby incorporated by reference in 15 ing or inhibiting spermatogenesis in a male subject. In their entirety . embodiments , the methods involve administering an effec tive amount of a compound or a salt thereof that inhibits a STATEMENT OF RIGHTS TO INVENTIONS bromodomain testis -specific protein (BRDT ) to the male MADE UNDER FEDERALLY SPONSORED subject. RESEARCH 20 In one aspect , the invention provides methods for reduc ing the rate ofmale fertility in a subject. In embodiments , the This work was supported by the following grant from the methods involve administering an effective amount of a National Institutes of Health , Grant No: K08CA128972 compound or a salt thereof that inhibits a BRDT to the male (Bradner ) . The government has certain rights in the inven subject. 25 In the above aspects , the methods involve administering tion . the compound or a salt thereof in an amount sufficient to reduce sperm number and / or reduce sperm motility . INCORPORATION BY REFERENCE OF In the above aspects , the methods involve administering MATERIAL IN ASCII TEXT FILE the compound or a salt thereof in an amount sufficient to induce azoospermia , oligozoospermia , and / or asthenozoo This application incorporates by reference the Sequence 30 spermia . In embodiments , the methods induce a contracep Listing containedned in the following ASCII texttext file being tive effect in the subject. submitted concurrently herewith : In aspect of the invention , the invention provides phar File name: TDQ - 00302 _ SEQLISTING . txt ; created Dec . maceutical compositions having a compound that inhibits 21, 2015, 30 KB in size . BRDT or a pharmaceutically acceptable salt or prodrug 35 thereof . In embodiments , the compound or a salt thereof is BACKGROUND OF THE INVENTION present in a amount effective to reduce or inhibit spermato genesis in a male subject. Although ~ 4 % of the mammalian genome encodes genes In embodiments , the compound or a salt thereof is present expressed in male germ cells during spermatogenesis , con - in an amount effective to reduce sperm number and /or traceptive drugs for men have remained elusive . To date , the 40 reduce sperm motility . only drugs in clinical trials are analogs that alter In embodiments , the compound or a salt thereof is present endogenous androgen production . This lack of contraceptive in a amount effective to induce azoospermia , oligozoosper alternatives for men is partially responsible for the high rate mia , and / or asthenozoospermia . In related embodiments , the compound or a salt thereof is present in a amount effective of unplanned pregnancies, especially in teenagersand , and finan the - 45 to induce a contraceptive effect in the subject . associated maternal mortality and ethical , social , and finan - 45 to Inind any of the above aspects , the compound or a salt thereof cial costs associated with abortions and deliveries to single is administered to the subject using any dosage and /or route mothers . To approach this dearth of contraceptive alterna of administration described herein . In embodiments , the tives for men , it is desirable to develop small molecules that compound or a salt thereof is administered to the subject could target spermatogenic - specific proteins that have been orally, transdermally , or by injection . In related embodi shown to be essential for both spermatogenesis and fertility 50 ments, the compound or a salt thereof is administered in the in mammals . One such contraceptive target is the bromodo - form of a tablet or capsule . In related embodiments , the main testis - specific protein , BRDT. compound or a salt thereof is administered by parenteral BRDT is a tissue - restricted , chromatin remodeling protein injection , intramuscular injection , intravenous injection , expressed in pachytene spermatocytes , diplotene spermato - subcutaneous implantation , subcutaneous injection , or trans cytes , and round spermatids. During post- meiotic matura - 55 dermal preparation . tion , BRDT localizes to the nucleus and reorganizes hyper In any of the above aspects , the compound or a salt thereof acetylated histones through twin acetyl- lysine recognition is administered in combination with a pharmaceutically modules , or bromodomains . The essential role of BRDT in acceptable carrier, excipient, or diluent. spermatogenesis is mediated by the first bromodomain In any of the above aspects , administration of the com ( BRDT ( 1 )) , which binds the tetra -acetylated amino -terminal 60 pound or a salt thereof reduces epididymal sperm number by tail of histone 4 (H4Kac4 ) with moderate potency ( 20 UM ) . at least about 25 % of the sperm number present in a control. Structural studies of murine BRDT have demonstrated that In embodiments , administration of the compound or a salt BRDT ( 1 ) binds a diacetylated histone 4 peptide thereof reduces epididymal sperm number by at least about (H4K5ac8ac ) in part through a conserved asparagine , akin to 10 % of the sperm number present in a control. In related seminal studies of other bromodomain co - activator proteins . 65 embodiments , only about 5 % of the spermatozoa remaining Genetic studies of BRDT have demonstrated that selective show progressive motility after administration of the com deletion of the BRDT ( 1 ) - encoding region is sufficient to pound or a salt thereof . US 9 , 789 ,120 B2 In any of the above aspects , administration of the com The term “ azoospermia ” refers to a spermatozoa content pound or a salt thereof lowers the spermatozoa concentration below 1 million per mL seminal fluid , approaching levels of to not more than 3 million /mL , 2 million /L , 1 million /mL , zero spermatozoa content, and are the result of suppression of spermatogenesis . 0 . 5 million /mL , 0 . 25 million -mL , or 0 . 1 million /mL . In 5 The term " oligozoospermia ” refers to a spermatozoa related embodiments , administration of the compound or a 5 content between 20 and one million per mL (mill / mL ) salt thereof lowers the spermatozoa concentration to not seminal fluid , and are the result of inhibited levels of more than 0 . 1 million /mL . spermatogenesis . In one aspect, the invention provides kits for reducing By “ bromodomain ” is meant a portion of a polypeptide male fertility . In embodiments , the kits contain an effective that recognizes acetylated lysine residues . In one embodi amount of a compound that inhibits BRDT or a pharmaceu - ment, a bromodomain of a BET family member polypeptide tically acceptable salt or prodrug thereof. In embodimentsinto , comprises approximately 110 amino acids and shares a the kits contain instructions for using the inhibitor in any of conserved fold comprising a left- handed bundle of four the methods described herein . alpha helices linked by diverse loop regions that interact with chromatin . In any of the above aspects, the compound is JQ1 or a 1a15 By “ BET family polypeptide” is meant a polypeptide compound of any of Formulas I -XXII , or any compound comprising two bromodomains and an extraterminal (ET ) disclosed herein , or a derivative or salt thereof. In embodi domain or a fragment thereof having transcriptional regu ments , the compound is JQ1 or a pharmaceutically accept latory activity or acetylated lysine binding activity . Exem able salt or prodrug thereof. plary BET family members include BRD2, BRD3, BRD4 In any of the above aspects, the compound or a salt thereof 20 and BRDT. is administered in combination with at least one additional By “ BRD2 polypeptide ” is meant a protein or fragment male contraceptive agent or device . In embodiments , the thereof having at least 85 % identity to NP _ 005095 that is additional male contraceptive is a condom . In embodiments, capable of binding chromatin or regulating transcription . the additional male contraceptive is a modulator of testos The sequence of an exemplary BRD2 polypeptide fol terone production , androgen receptor function or stability . 25 lows: Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description , or may be learned by MLQNVTPHNKLPGEGNAGLLGLGPEAAAPGKRIRKPSLLYEGFESPTMAS practice of the invention . The objects and advantages of the VPALQLTPANPPPPEVSNPKKPGRVTNQLQYLHKVVMKALWKHQFAWPFR invention will be realized and attained by means of the 30 elements and combinations disclosed herein , including those QPVDAVKLGLPDYHKIIKQPMDMGTI KRRLENNYYWAASECMODFNTMFT pointed out in the appended claims. It is to be understood that both the foregoing general description and the following NCYIYNKPTDDIVLMAQTLEKIFLQKVASMPQEEQELVVTIPKNSHKKGA detailed description are exemplary and explanatory only and KLAALQGSVTSAHQVPAVSSVSHTALYTPPPEIPTTVLNIPHPSVISSPL are not restrictive of the invention as claimed . The accom - 35 panying drawings, which are incorporated in and constitute LKSLHSAGPPLLAVTAAPPAQPLAKKKGVKRKADTTTPTPTAILAPGSPA a part of this specification , illustrate several embodiments of the invention and , together with the description , serve to SPPGSLEPKAARLPPMRRESGRPIKPPRKDLPDSQQQHQSSKKGKLSEQL explain the principles of the invention . KHCNGILKELLSKKHAAYAWPFYKPVDASALGLHDYHDIIKHPMDLSTVK 40 Definitions RKMENRDYRDAQEFAADVRLMFSNCYKYNPPDHDWVAMARKLQDVFEFRY To facilitate an understanding of the present invention , a AKMPDEPLBPGPLPVSTAMPPGLAKSSSESSSSESSSESSSEEEEEEDEE number of terms and phrases are defined below . DEEEEESESSDSEEERAHRLAELQEQLRAVHEQLAALSQGPISKPKRKRE refersThe toterm lowering reducing the oramount inhibiting of spermatozoaspermatozoa presentemission in 45 KKEKKKKRKAEKHRGRAGADEDDKGPRAPRPPOPKKSKKASGSGGCSAAL seminal fluid during discharge of the seminal fluid from a GPSGFGPSGGSGTKLPKKATKTAPPALPTGYDSEEEEESRPMSYDEKROL male subject. Reduction or inhibition of spermatozoa levels in seminal fluid can be effected by suppressing spermato SLDINKLPGEKLGRVVHIIQAREPSLRDSNPEEIEIDFETLKPSTLRELE genesis , inducing azoospermia , inducing oligozoospermia , 50 RYVLSCLRKKPRKPYTIKKPVGKTKBELALEKKRELEKRLODVSGOLNST and the like . Thus, in the context of the present invention , " reducing or inhibiting spermatozoa emission ” has the effect KKPPKKANEKTESSSAQQVAVSRLSASSSSSSSSSSSSSSSSDTSDSDS of inhibiting and/ or reducing the rate of fertilization when aG the discharged seminal fluid contacts ova from a female subject. 55 By “ BRD2 nucleic acid molecule ” is meant a polynucle " Spermatogenesis ” refers to the overall process of game- otide encoding a BRD2 polypeptide or fragment thereof. togenesis in the male . Spermatogenesis takes place in the By “ BRD3 polypeptide” is meant a protein or fragment seminiferous tubule and is directly regulated by levels of thereof having at least 85 % identity to NP _ 031397 . 1 that is follicle stimulating hormone and androgen at the periphery capable of binding chromatin or regulating transcription . of the seminiferous tubule , particularly upon the Sertoli The sequence of an exemplary BRD3 polypeptide fol cells . lows: 1 mstattvapa gipatpgpvn ppppevsnps kpqrktnqlq ymqnvvvkti wkhqfawpfy 61 qpvdaikinl pdyhkiiknp mdmgtikkri ennyywsase cmqdfntmft ncyiynkptd US 9 ,789 , 120 B2

121 divlmaqale kiflqkyaqm pqeevellpp- continued apkgkgrkpa agaqsagtqq vaayssvspa 181 tpfqsvpptv sctpviaatp vptitanvts vpvppaaapp ppatpivpvv pptppvvkkk 241 gvkrkadttt pttsaitasr sesppplsdp kakvvarre sggrpikppk kdledgevpq 301 hagkkgklse hlrycdsilr emlskkhaay awpfykpvda ealelhdyhd iikhpmdlst 361 vkrkmdgrey pdaggfaadv rlmfsncyky nppdhevvam arklqdvfem rfakmpdepv 421 eapalpapaa pmvakgaess rsseesssds gssdseeera trlaelqeqi kavheqiaal 481 sqapvnkpkk kkekkekekk kkdkekekek hkvkaeeekk akvappakqa qqkkapakka 541 nstttagrqi kkggkqasas ydseeeeegl pmsydekrql sldinrlpge klgrvvhiiq 601 srepslrdsn pdeieidfet lkpttlrele ryvksclqkk qrkpfsasgk kqaskskeel 661 agekkkelek riqdvsgqls sskkparkek pgsapsggps rlsssssses gsssssgsss 721 dssdee By “ Brd3 nucleic acid molecule” is meant a polynucle - 20 otide encoding a BRD3 polypeptide. By “ BRD4 polypeptide” is meant a protein or fragment thereof having at least 85 % identity to NP _ 055114 that is capable of binding chromatin or regulating transcription . 1 msaesgpgtr lrnlpvmgdg let sqmsttq aqaqpqpana astnppppet snpnkpkrqt 61 nqlqyllrvv lktlwkhqfa wpfqqpvdav klnlpdyyki iktpmdmgti kkrlennyyw 121 naqeciqdfn tmftncyiyn kpgddivlma ealeklflqk inelpteete imivqakgrg 181 rgrketgtak pgvstvpntt qastppqtqt pqpnpppvqa tphpfpavtp flivqtpvmt 241 vippqplqtp ppvppppp papapqpvqs hppiiaatpq pvktkkgvkr kadtttptti 301 dpiheppslp pepkttklgq rreserpvkp pkkdvpdsqq hpapeksskv seqlkccsgi 361 lkemfakkha ayawpfykpv dvealglhdy cdiikhpmdm stiksklear eyrdaqefga 421 dvrlmfsncy kynppdhevv amarklqdvf emrfakmpde peepvvavss pavppptkvv 481 appsssdsss dsssdsdsst ddseeeraqr laelqeqlka vheqlaalsq pqqnkpkkke 541 kdkkekkkek hkrkeeveen kkskakeppp kktkknnssn snvskkepap mkskppptye 601 Seeedkckpm syeekrqlsl dinklpgekl grvvhiiqsr epslknsnpd eieidfetlk 661 pstirelery vtsclrkkrk pgaekvdvia qsskmkqfss sesesssess ssdsedseta 721 pa By “ Brd4 nucleic acid molecule ” is meant a polynucle otide that encodes a BRD4 polypeptide . By “ BRDT polypeptide is meant a protein or fragment 50 thereof having at least 85 % identity to NP _ 001717 that is capable of binding chromatin or regulating transcription . 1 mslpsrqtai ivnppppeyi ntkkngrltn qlqylqkvvl kdlwkhsfsw pfqrpvdavk 61 iqlpdyytii knpmdintik krlenkyyak aseciedfnt mfsncylynk pgddivlmaq 121 aleklfmqkl sqmpqeeqvv gvkerikkgt qqniavssak eksspsatek vfkqqeipsy 181 fpktsispin vvwgasvnss sqtaaqvtkg vkrdadtttp atsavkasse fsptfteksv 241 alppikenmp knvlpdsqqq ynvvktvkvt eglrhcseil kemlakkhfs yawpfynpvd 301 vnalglhnyy dvvknpmdlg tikekmdnqe ykdaykfaad vrlmfmncyk ynppdhevyt 361 marmlqdvfe thfskipiep vesmplcyik tditettgre ntneassegn ssddsederv 421 krlaklqeql kavhqqlqvi sqvpfrkink kkekskkekk kekvnnsnen prkmceqmrl US 9 ,789 , 120 B2

- continued 481 kekskrnqpk krkqqfiglk sedednakpm nydekrqlsl ninklpgdkl grvvhiiqsr 541 epslsnsnpd eieidfetlk astireleky vsaclrkrpl kppakkimms keelhsqkkq 601 elekrildvn nqlnsrkrat ksdktqpska venvsrlses sssssssses essssdlass 661 dssdsesemf pkftevkond spskenvkkm knecilpegr tgvtqigyev qdttsantti 721 vhqttpshvm ppnhhqlafn yqelehlqtv knisplqilp psgdseqlsn gitmvhpsgd 781 sdttmlesec qapvqkdiki knadswkslg kpvkpsgvmk ssdelfnqfr kaaiekevka 841 rtgelirkhl eqntkelkas qenqrdlgng ltvesfsnki qnkcsgeeqk ehqqsseaqd 901 ksklwllkdr dlarqkeqer rrreamvgti dmtlqsdimt mfennfd 1515 By “ BRDT nucleic acid molecule ” is meant a polynucle - ylhexyl, 2 , 2 - diethylhexyl , 3 , 3 - diethylhexyl and the like . otide encoding a BRDT polypeptide. Alkyl groups included in compounds of this invention may “ Administering ” is defined herein as a means of providing be unsubstituted , or optionally substituted with one or more an agent to a subject in a manner that results in the agent substituents , such as amino , alkylamino , arylamino , het being inside the subject' s body . Such an administration can e roarylamino , alkoxy , alkylthio , oxo , halo , acyl, nitro , be by any route including , without limitation , oral, trans- hydroxyl, cyano, aryl, heteroaryl, alkylaryl, alkylheteroaryl, dermal, mucosal ( e . g . , vagina , rectum , oral , or nasal aryloxy , heteroaryloxy, arylthio , heteroarylthio , arylamino , mucosa ), by injection (e . g. , subcutaneous, intravenous, par - heteroarylamino , carbocyclyl, carbocyclyloxy, carbocyclyl enterally , intraperitoneally , intrathecal) , or by inhalation 25 thio , carbocyclylamino , heterocyclyl, heterocyclyloxy , het ( e. g ., oral or nasal ). Pharmaceutical preparations are given erocyclylamino , heterocyclylthio , and the like . Lower alkyls by forms suitable for the desired route of administration . are typically preferred for the compounds of this invention . By " agent” or “ compound ” is meant any small molecule As used herein , the term an " aromatic ring " or " aryl ” chemical compound , antibody , nucleic acid molecule, or means a monocyclic or polycyclic - aromatic ring or ring polypeptide , or fragments thereof . 30 radical comprising carbon and hydrogen atoms. Examples of By " ameliorate ” is meant decrease , suppress , attenuate , suitable aryl groups include , but are not limited to , phenyl, diminish , arrest , or stabilize the development or progression tolyl, anthacenyl, fluorenyl, indenyl, azulenyl, and naphthyl, of a disease . as well as benzo - fused carbocyclic moieties such as 5 , 6 , 7 , By “ alteration ” is meant a change ( increase or decrease ) 8 -tetrahydronaphthyl . An aryl group can be unsubstituted or in the expression levels or activity of a gene or polypeptide 35 optionally is substituted with one or more substituents , e . g . , as detected by standard art known methods such as those substituents as described herein for alkyl groups ( including described herein . As used herein , an alteration includes a without limitation alkyl (preferably , lower alkyl or alkyl 10 % change in expression levels , preferably a 25 % change , substituted with one or more halo ), hydroxy, alkoxy (pref more preferably a 40 % change , and most preferably a 50 % erably , lower alkoxy ) , alkylthio , cyano , halo , amino , boronic or greater change in expression levels . 40 acid ( B (OH ) 2 , and nitro ) . In certain embodiments , the aryl By “ analog” is meant a molecule that is not identical, but group is a monocyclic ring , wherein the ring comprises 6 has analogous functional or structural features . For example , carbon atoms. a polypeptide analog retains at least some of the biological The term " diastereomers ” refers to stereoisomers with activity of a corresponding naturally - occurring polypeptide , two or more centers of dissymmetry and whose molecules while having certain biochemical modifications that enhance 45 are not mirror images of one another. the analog ' s function relative to a naturally occurring poly . The term " enantiomers ” refers to two stereoisomers of a peptide . Such biochemical modifications could increase the compound which are non - superimposable mirror images of analog ' s protease resistance , membrane permeability , or one another . An equimolar mixture of two enantiomers is half- life , without altering , for example , ligand binding . An called a “ racemic mixture ” or a “ racemate . ” analog may include an unnatural amino acid . 50 The term “ halogen ” designates – F , C1, - Br or — I . As used herein , the term “ alkyl” means a saturated The term " haloalkyl” is intended to include alkyl groups straight chain or branched non - cyclic hydrocarbon typically as defined above that are mono - , di- or polysubstituted by having from 1 to 10 carbon atoms. Representative saturated halogen , e . g ., fluoromethyl and trifluoromethyl. straight chain alkyls include methyl, ethyl, n -propyl , n -butyl , The term “ hydroxyl ” means – OH . n - pentyl, n -hexyl , n -heptyl , n -octyl , n - nonyl and n -decyl ; 55 The term " heteroatom ” as used herein means an atom of while saturated branched alkyls include isopropyl, sec - butyl, any element other than carbon or hydrogen . Preferred het isobutyl, tert- butyl , isopentyl, 2 -methylbutyl , 3 -methylbutyl , eroatoms are nitrogen , oxygen , sulfur and phosphorus . 2 -methylpentyl , 3 -methylpentyl , 4 -methylpentyl , 2 -methyl The term " heteroaryl” refers to an aromatic 5 - 8 membered hexyl, 3 -methylhexyl , 4 -methylhexyl , 5 -methylhexyl , 2 , 3 - monocyclic, 8 - 12 membered bicyclic , or 11 - 14 membered dimethylbutyl, 2 , 3 - dimethylpentyl, 2 ,4 -dimethylpentyl , 2 , 3 - 60 tricycle ring system having 1- 4 ring heteroatoms if mono dimethylhexyl, 2 ,4 -dimethylhexyl , 2 ,5 -dimethylhexyl , 2 , 2 cyclic , 1 -6 heteroatoms if bicyclic , or 1 - 9 heteroatoms if dimethylpentyl , 2 , 2 -dimethylhexyl , 3 , 3 -dimethylpenty1 , 3 , 3 - tricyclic , said heteroatoms selected from O , N , or S , and the dimethylhexyl, 4 , 4 - dimethylhexyl, 2 -ethylpentyl , remainder ring atoms being carbon . Heteroaryl groups may 3 -ethylpentyl , 2 - ethylhexyl, 3 - ethylhexyl, 4 -ethylhexyl , be optionally substituted with one or more substituents , e . g ., 2 -methyl - 2 - ethylpentyl, 2 -methyl - 3 - ethylpentyl, 2 -methyl - 65 substituents as described herein for aryl groups. Examples of 4 - ethylpentyl, 2 -methyl - 2 - ethylhexyl, 2 -methyl - 3 - ethyl- heteroaryl groups include , but are not limited to , pyridyl, hexyl, 2 -methyl - 4 - ethylhexyl, 2, 2 -diethylpentyl , 3, 3 - dieth furanyl, benzodioxolyl, thienyl, pyrrolyl , oxazolyl , oxadiaz US 9 ,789 , 120 B2 10 olyl, imidazolyl , thiazolyl, isoxazolyl, quinolinyl, pyrazolyl , so long as basic or novel characteristics of that which is isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, recited is not changed by the presence of more than that triazolyl, thiadiazolyl, isoquinolinyl , indazolyl, benzox - which is recited , but excludes prior art embodiments. azolyl , benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl, “ Detect ” refers to identifying the presence, absence or benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzox - 5 amount of the analyte to be detected adiazolyl, and indolyl. By “ detectable label” is meant a composition that when The term " heterocyclic” as used herein , refers to organic linked to a molecule of interest renders the latter detectable , compounds that contain at least at least one atom other than carbon ( e . g . , S , O , N ) within a ring structure . The ring via spectroscopic , photochemical , biochemical, immuno structure in these organic compounds can be either aromatic 10 chemical, or chemical means . For example , useful labels or, in certain embodiments , non -aromatic . Some examples 10 include radioactive isotopes , magnetic beads , metallic of heterocyclic moieties include, are not limited to , pyridine , beads, colloidal particles , fluorescent dyes, electron -dense pyrimidine , pyrrolidine , furan , tetrahydrofuran , tetrahydro reagents , enzymes ( for example , as commonly used in an thiophene , and dioxane . ELISA ) , biotin , digoxigenin , or haptens . The term “ isomers ” or “ stereoisomers ” refers to com - 15 ByE " effective amount” is meant the amount of an agent pounds which have identical chemical constitution , but required to provide contraception to an otherwise fertile differ with regard to the arrangement of the atoms or groups male . The effective amount of active compound ( s ) used to in space . practice the present invention for therapeutic treatment of a The term “ isotopic derivatives” includes derivatives of disease varies depending upon the manner of administration , compounds in which one or more atoms in the compounds 20 the age , body weight, and general health of the subject. are replaced with corresponding isotopes of the atoms. For Ultimately , the attending physician or veterinarian will example , an isotopic derivative of a compound containing a decide the appropriate amount and dosage regimen . Such carbon atom (C12 ) would be one in which the carbon atom amount is referred to as an " effective” amount. of the compound is replaced with the C13 isotope . The term " enantiomers ” refers to two stereoisomers of a By " computer modeling” is meant the application of a 25 compound which are non - superimposable mirror images of computational program to determine one or more of the one another. An equimolar mixture of two enantiomers is following : the location and binding proximity of a ligand to called a “ racemic mixture ” or a " racemate .” a binding moiety , the occupied space of a bound ligand , the The term “ halogen ” designates — F , C1, Br or — I . amount of complementary contact surface between a bind - The term “ haloalkyl” is intended to include alkyl groups ing moiety and a ligand , the deformation energy of binding 30 as defined above that are mono -, di- or polysubstituted by of a given ligand to a binding moiety , and some estimate of halogen , e . g ., fluoromethyl and trifluoromethyl. hydrogen bonding strength , van der Waals interaction , The term “ hydroxyl” means OH . hydrophobic interaction , and / or electrostatic interaction The term " heteroatom ” as used herein means an atom of energies between ligand and binding moiety . Computer any element other than carbon or hydrogen . Preferred het modeling can also provide comparisons between the features 35 eroatoms are nitrogen , oxygen , sulfur and phosphorus . of a model system and a candidate compound . For example , The term " heteroaryl” refers to an aromatic 5 - 8 membered a computer modeling experiment can compare a pharma monocyclic , 8 - 12 membered bicyclic , or 11 - 14 membered cophore model of the invention with a candidate compound tricyclic ring system having 1 - 4 ring heteroatoms if mono to assess the fit of the candidate compound with the model . cyclic , 1 - 6 heteroatoms if bicyclic , or 1 - 9 heteroatoms if By a " computer system ” is meant the hardware means , 40 tricyclic , said heteroatoms selected from O , N , or S , and the software means and data storage means used to analyse remainder ring atoms being carbon . Heteroaryl groups may atomic coordinate data . The minimum hardware means of be optionally substituted with one or more substituents . the computer- based systems of the present invention com - Examples of heteroaryl groups include , but are not limited prises a central processing unit (CPU ) , input means , output to , pyridyl , furanyl, benzodioxolyl, thienyl, pyrrolyl , means and data storage means . Desirably a monitor is 45 oxazolyl , oxadiazolyl, imidazolyl thiazolyl, isoxazolyl, qui provided to visualize structure data . The data storage means nolinyl, pyrazolyl, isothiazolyl , pyridazinyl , pyrimidinyl, may be RAM or means for accessing computer readable pyrazinyl, triazinyl, triazolyl, thiadiazolyl, isoquinolinyl, media of the invention . Examples of such systems are indazolyl, benzoxazolyl, benzofuryl , indolizinyl, imida microcomputer workstations available from Silicon Graph - zopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, ben ics Incorporated and Sun Microsystems running Unix based , 50 zothiadiazolyl, benzoxadiazolyl, and indolyl. Windows NT or IBM OS / 2 operating systems. The term " heterocyclic ” as used herein , refers to organic By “ computer readable media " is meant any media which compounds that contain at least at least one atom other than can be read and accessed directly by a computer e . g . so that carbon ( e . g . , S , O , N ) within a ring structure . The ring the media is suitable for use in the above - mentioned com - structure in these organic compounds can be either aromatic puter system . The media include , but are not limited to : 55 or non - aromatic . Some examples of heterocyclic moieties magnetic storage media such as floppy discs, hard disc include , are not limited to , pyridine , pyrimidine , pyrrolidine , storage medium and magnetic tape; optical storage media furan , tetrahydrofuran , tetrahydrothiophene, and dioxane . such as optical discs or CD - ROM ; electrical storage media The term “ isomers” or “ stereoisomers ” refers to com such as RAM and ROM ; and hybrids of these categories pounds which have identical chemical constitution , but such as magnetic /optical storage media . 60 differ with regard to the arrangement of the atoms or groups In this disclosure, " comprises, " " comprising , " " contain - in space . ing ” and “ having” and the like can have the meaning The term “ isotopic derivatives” includes derivatives of ascribed to them in U . S . patent law and can mean compounds in which one or more atoms in the compounds “ includes, ” “ including , " and the like ; " consisting essentially are replaced with corresponding isotopes of the atoms. For of” or “ consists essentially ” likewise has the meaning 65 example , an isotopic derivative of a compound containing a ascribed in U . S . patent law and the term is open - ended , carbon atom ( C + 2 ) would be one in which the carbon atom allowing for the presence of more than that which is recited of the compound is replaced with the C isotope . US 9 , 789 ,120 B2 12 The invention provides a number of targets that are useful The term “ optical isomers ” as used herein includes mol for the development of highly specific drugs to reduce ecules, also known as chiral molecules , that are exact fertility in a male subject. In addition , the methods of the non - superimposable mirror images of one another . invention provide a facile means to identify other contra The phrases " parenteral administration ” and “ adminis ceptive therapies that are safe for use in male subjects . In 5 tered parenterally ” as used herein means modes of admin addition , the methods of the invention provide a route for istration other than enteral and topical administration , usu analyzing virtually any number of compounds for effects on ally by injection , and includes : without limitation , a disease described herein with high - volume throughput, intravenous , intramuscular, intraarterial, intrathecal, intraca high sensitivity , and low complexity . psular, intraorbital, intracardiac, intradermal, intraperito : 10 neal, transtracheal, subcutaneous , subcutiular, intraarticu By “ fitting ” is meant determining by automatic , or semi- 10 lare , subcapsular, subarachnoid , intraspinal and intrasternal automatic means, interactions between one or more atomsof injection and infusion . an agent molecule and one or more atoms or binding sites of “ Pharmaceutically acceptable ” refers to approved or a BET family member ( e . g . , a bromodomain BRD2, BRD3 , approvable by a regulatory agency of the Federal or a state BRD4 and BRDT) , and determining the extent to whichh 15 government or listed in the U . S . Pharmacopeia or other such interactions are stable . Various computer -based meth generally recognized pharmacopeia for use in animals , ods for fitting are described further herein . including humans. By “ fragment” is meant a portion of a polypeptide or “ Pharmaceutically acceptable excipient, carrier or adju nucleic acid molecule . This portion contains, preferably , at vant” refers to an excipient, carrier or adjuvant that can be least 10 % , 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , or 90 % 20 administered to a subject, together with an agent , e . g ., any of the entire length of the reference nucleic acid molecule or of the compounds described herein , and which does not polypeptide. A fragment may contain 10 , 20 , 30 , 40 , 50 , 60 , destroy the pharmacological activity thereof and is nontoxic 70 , 80 , 90 , or 100 , 200 , 300 , 400 , 500 , 600 , 700 , 800 , 900 , when administered in doses sufficient to deliver a therapeu or 1000 nucleotides or amino acids. tic amount of the agent. “ Hybridization ” means hydrogen bonding , which may be 25 The terms " polycyclyl” or “ polycyclic radical ” refer to the Watson -Crick , Hoogsteen or reversed Hoogsteen hydrogen radical of two or more cyclic rings ( e . g ., cycloalkyls , bonding , between complementary nucleobases. For cycloalkenyls , cycloalkynyls , aryls and /or heterocyclyls ) in example , adenine and thymine are complementary nucle - which two or more carbons are common to two adjoining obases that pair through the formation of hydrogen bonds. rings , e . g . , the rings are “ fused rings” . Rings that are joined By “ isolated polynucleotide ” is meant a nucleic acid ( e . g ., 30 through non -adjacent atoms are termed " bridged ” rings . a DNA ) that is free of the genes which , in the naturally . Each of the rings of the polycycle can be substituted with occurring genome of the organism from which the nucleic such substituents as described above, as for example , halo acid molecule of the invention is derived , flank the gene . The gen , hydroxyl, alkylcarbonyloxy , arylcarbonyloxy , alkoxy term therefore includes, for example, a recombinant DNA carbonyloxy , aryloxycarbonyloxy, carboxylate , alkylcarba that is incorporated into a vector , into an autonomously 35 nyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, replicating plasmid or virus; or into the genomic DNA of a alkoxyl, phosphate, phosphonato , phosphinato , cyano , prokaryote or eukaryote ; or that exists as a separate mol amino ( including alkyl amino , dialkylamino , arylamino , ecule ( for example , a cDNA or a genomic or cDNA fragment diarylamino , and alkylarylamino ) , acylamino ( including produced by PCR or restriction endonuclease digestion ) alkylcarbonylamino , arylcarbonylamino , carbamoyl and independent of other sequences . In addition , the term 40 ureido ), amidino , imino , alkylthio , arylthio , thiocarboxylate , includes an RNA molecule that is transcribed from a DNA sulfates , sulfonato , sulfamoyl, sulfonamido , nitro , trifluo molecule , as well as a recombinant DNA that is part of a romethyl, cyano, azido , heterocyclyl , alkyl, alkylaryl , or an hybrid gene encoding additional polypeptide sequence . aromatic or heteroaromatic moiety . By an “ isolated polypeptide” is meant a polypeptide of the The term “ polymorph ” as used herein , refers to solid invention that has been separated from components that 45 crystalline forms of a compound of the present invention or naturally accompany it . Typically , the polypeptide is isolated complex thereof. Different polymorphs of the same com when it is at least 60 % , by weight, free from the proteins and pound can exhibit different physical, chemical and /or spec naturally - occurring organic molecules with which it is natu - troscopic properties . Different physical properties include , rally associated . Preferably , the preparation is at least 75 % , but are not limited to stability ( e . g ., to heat or light) , more preferably at least 90 % , and most preferably at least 50 compressibility and density ( important in formulation and 99 % , by weight, a polypeptide of the invention . An isolated product manufacturing ) , and dissolution rates (which can polypeptide of the invention may be obtained , for example , affect bioavailability ). Differences in stability can result by extraction from a natural source , by expression of a from changes in chemical reactivity ( e . g . , differential oxi recombinant nucleic acid encoding such a polypeptide; or by dation , such that a dosage form discolors more rapidly when chemically synthesizing the protein . Purity can be measured 55 comprised of one polymorph than when comprised of by any appropriate method , for example , column chroma - another polymorph ) or mechanical characteristics ( e . g . , tab tography, polyacrylamide gel electrophoresis , or by HPLC lets crumble on storage as a kinetically favored polymorph analysis . converts to thermodynamically more stable polymorph ) or By “ marker ” is meant any protein or polynucleotide both ( e . g ., tablets of one polymorph are more susceptible to having an alteration in expression level or activity that is 60 breakdown at high humidity ) . Different physical properties associated with a disease or disorder. of polymorphs can affect their processing . As used herein , “ obtaining ” as in “ obtaining an agent” The term “ prodrug ” includes compounds with moieties includes synthesizing , purchasing , or otherwise acquiring which can be metabolized in vivo . Generally , the prodrugs the agent. are metabolized in vivo by esterases or by other mechanisms The term “ obtaining ” as in “ obtaining compound ” is 65 to active drugs. Examples of prodrugs and their uses are intended to include purchasing , synthesizing or otherwise well -known in the art (See , e . g . , Berge et al. ( 1977 ) “ Phar acquiring the compound . maceutical Salts” , J . Pharm . Sci. 66 : 1 - 19 ) . The prodrugs can US 9 ,789 , 120 B2 13 14 be prepared in situ during the final isolation and purification thereof. Such nucleic acid molecules need not be 100 % of the compounds, or by separately reacting the purified identical with an endogenous nucleic acid sequence, but will compound in its free acid form or hydroxyl with a suitable typically exhibit substantial identity . Polynucleotides having esterifying agent . Hydroxyl groups can be converted into “ substantial identity ” to an endogenous sequence are typi esters via treatment with a carboxylic acid . Examples of 5 cally capable of hybridizing with at least one strand of a prodrug moieties include substituted and unsubstituted , double -stranded nucleic acid molecule . By “ hybridize” is branch or unbranched lower alkyl ester moieties, ( e . g ., meant pair to form a double -stranded molecule between propionoic acid esters) , lower alkenyl esters , di- lower alkyl complementary polynucleotide sequences ( e . g . , a gene amino lower -alkyl esters ( e . g ., dimethylaminoethyl ester ), described herein ), or portions thereof, under various condi acylamino lower alkyl esters ( e . g . , acetyloxymethyl ester ), 10 tions of stringency . (See , e . g ., Wahl, G . M . and S . L . Berger acyloxy lower alkyl esters (e . g. , pivaloyloxymethyl ester ), (1987 ) Methods Enzymol. 152 : 399 ; Kimmel, A . R . ( 1987 ) aryl esters (phenyl ester ), aryl -lower alkyl esters ( e. g ., Methods Enzymol. 152 :507 ) . benzyl ester ), substituted ( e .g ., with methyl, halo , or For example , stringent salt concentration will ordinarily methoxy substituents ) aryl and aryl- lower alkyl esters , be less than about 750 mM NaCl and 75 mM trisodium amides , lower alkyl amides, di- lower alkyl amides , and 15 citrate , preferably less than about 500 mM NaCl and 50 mM hydroxy amides . Preferred prodrug moieties are propionoic trisodium citrate , and more preferably less than about 250 acid esters and acyl esters . Prodrugs which are converted to mM NaCl and 25 mM trisodium citrate . Low stringency active forms through other mechanisms in vivo are also hybridization can be obtained in the absence of organic included . solvent, e . g ., formamide, while high stringency hybridiza Furthermore the indication of stereochemistry across a 20 tion can be obtained in the presence of at least about 35 % carbon - carbon double bond is also opposite from the general formamide , and more preferably at least about 50 % forma chemical field in that “ Z ” refers to what is often referred to mide . Stringent temperature conditions will ordinarily as a " cis ” ( same side ) conformation whereas “ E ” refers to include temperatures of at least about 30° C ., more prefer what is often referred to as a " trans ” (opposite side ) con - ably of at least about 37° C . , and most preferably of at least formation . Both configurations , cis / trans and / or Z / E are 25 about 42° C . Varying additional parameters , such as hybrid encompassed by the compounds of the present invention . ization time, the concentration of detergent, e . g . , sodium With respect to the nomenclature of a chiral center, the dodecyl sulfate (SDS ) , and the inclusion or exclusion of terms“ d ” and “ l ” configuration are as defined by the IUPAC carrier DNA , are well- known to those skilled in the art . Recommendations. As to the use of the terms, diastereomer , Various levels of stringency are accomplished by combining racemate , epimer and enantiomer, these will be used in their 30 these various conditions as needed . In a preferred embodi normal context to describe the stereochemistry of prepara ment, hybridization will occur at 30° C . in 750 mM NaCl, tions . 75 mM trisodium citrate , and 1 % SDS . In a more preferred By " reduces ” is meant a negative alteration of at least embodiment, hybridization will occur at 37° C . in 500 mM 10 % , 25 % , 50 % , 75 % , or 100 % . NaC1, 50 mM trisodium citrate , 1 % SDS , 35 % formamide , By “ reference ” is meant a standard or control condition . 35 and 100 ug/ ml denatured salmon sperm DNA (ssDNA ). In A “ reference sequence” is a defined sequence used as a a most preferred embodiment, hybridization will occur at basis for sequence comparison . A reference sequence may be 42° C . in 250 mM NaCl, 25 mM trisodium citrate , 1 % SDS , a subset of or the entirety of a specified sequence ; for 50 % formamide , and 200 ug /ml ssDNA . Useful variations example, a segment of a full - length cDNA , or gene on these conditions will be readily apparent to those skilled sequence, or the complete cDNA or gene sequence. For 40 in the art . polypeptides, the length of the reference polypeptide For most applications , washing steps that follow hybrid sequence will generally be at least about 16 amino acids, ization will also vary in stringency . Wash stringency condi preferably at least about 20 amino acids , more preferably at tions can be defined by salt concentration and by tempera least about 25 amino acids, and even more preferably about ture . As above, wash stringency can be increased by 35 amino acids , about 50 amino acids, or about 100 amino 45 decreasing salt concentration or by increasing temperature . acids . For nucleic acids , the length of the reference nucleic For example , stringent salt concentration for the wash steps acid sequence will generally be at least about 50 nucleotides , will preferably be less than about 30 mM NaCl and 3 mM preferably at least about 60 nucleotides, more preferably at trisodium citrate, and most preferably less than about 15 mM least about 75 nucleotides , and even more preferably about NaCl and 1. 5 mM trisodium citrate . Stringent temperature 100 nucleotides or about 300 nucleotides or any integer 50 conditions for the wash steps will ordinarily include a thereabout or therebetween . temperature of at least about 25° C . , more preferably of at By " specifically binds ” is meant a compound or antibody least about 42° C . , and even more preferably of at least about that recognizes and binds a polypeptide of the invention , but 68° C . In a preferred embodiment, wash steps will occur at which does not substantially recognize and bind other mol- 25° C . in 30 mM NaC1, 3 mM trisodium citrate , and 0 . 1 % ecules in a sample , for example , a biological sample , which 55 SDS . In a more preferred embodiment, wash steps will occur naturally includes a polypeptide of the invention . at 42 C in 15 mM NaC1, 1. 5 mM trisodium citrate , and 0 . 1 % Nucleic acid molecules useful in the methods of the SDS . In a more preferred embodiment, wash steps will occur invention include any nucleic acid molecule that encodes a at 68° C . in 15 mM NaCl, 1 . 5 mM trisodium citrate , and polypeptide of the invention or a fragment thereof. Such 0 . 1 % SDS . Additional variations on these conditions will be nucleic acid molecules need not be 100 % identical with an 60 readily apparent to those skilled in the art . Hybridization endogenous nucleic acid sequence , but will typically exhibit techniques are well - known to those skilled in the art and are substantial identity . Polynucleotides having " substantial described , for example , in Benton and Davis ( Science 196 : identity ” to an endogenous sequence are typically capable of 180 , 1977 ); Grunstein and Hogness (Proc . Natl. Acad . Sci. , hybridizing with at least one strand of a double -stranded USA 72 : 3961 , 1975 ) ; Ausubel et al. (Current Protocols in nucleic acid molecule . Nucleic acid molecules useful in the 65 Molecular Biology, Wiley Interscience , New York , 2001) ; methods of the invention include any nucleic acid molecule Berger and Kimmel (Guide to Molecular Cloning Tech that encodes a polypeptide of the invention or a fragment niques, 1987 , Academic Press, New York ) ; and Sambrook et US 9 ,789 , 120 B2 15 16 al. , Molecular Cloning: A Laboratory Manual , Cold Spring As used herein , the terms “ treat ,” “ treating, " " treatment, ” Harbor Laboratory Press , New York . and the like refer to reducing or ameliorating a disorder By “ substantially identical ” is meant a polypeptide or and /or symptoms associated therewith . By “ ameliorate ” is nucleic acid molecule exhibiting at least 85 % identity to a meant decrease , suppress, attenuate , diminish , arrest , or reference amino acid sequence ( for example , any one of the 5 stabilize the development or progression of a disease. It will amino acid sequences described herein ) or nucleic acid be appreciated that, although not precluded , treating a dis sequence ( for example , any one of the nucleic acid order or condition does not require that the disorder , con sequences described herein ) . Preferably , such a sequence is dition or symptoms associated therewith be completely eliminated . at least 85 % , 90 % , 95 % , 99 % or even 100 % identical at theor 10 As used herein , the terms " prevent, " " preventing ,” “ pre amino acid level or nucleic acid to the sequence used for vention , " " prophylactic treatment " and the like refer to comparison . reducing the probability of developing a disorder or condi Sequence identity is typically measured using sequence tion in a subject , who does not have , but is at risk of or analysis software ( for example , Sequence Analysis Software susceptible to developing a disorder or condition . Package of the Genetics Computer Group , University of 15 “ An effective amount ” refers to an amount of a com Wisconsin Biotechnology Center , 1710 University Avenue , pound , which confers a contraceptive effect on the treated Madison , Wis . 53705, BLAST, BESTFIT, GAP, or PILEUP/ subject. The effect may be objective ( i . e . , measurable by PRETTYBOX programs) . Such software matches identical some test or marker ) or subjective ( i. e . , subject gives an or similar sequences by assigning degrees of homology to indication of or feels an effect ). An effective amount of a various substitutions, deletions, and /or other modifications . 20 compound described herein may range from about 1 mg/ Kg Conservative substitutions typically include substitutions to about 5000 mg/ Kg body weight. Effective doses will also within the following groups: glycine , alanine; valine , iso - vary depending on route of administration , as well as the leucine, leucine ; aspartic acid , glutamic acid , asparagine , possibility of co -usage with other agents . In embodiments of glutamine ; serine , threonine ; lysine, arginine ; and phenyl- the present invention , " an effective amount” of an agent or alanine , tyrosine . In an exemplary approach to determining 25 composition is an amount sufficient to effect contraception . the degree of identity , a BLAST program may be used , with Ranges provided herein are understood to be shorthand a probability score between e .sup . -3 and e .sup .- 100 indicat - for all of the values within the range. For example , a range ing a closely related sequence . of 1 to 50 is understood to include any number, combination By “ reduces” or “ increases” is meant a negative or of numbers , or sub -range from the group consisting of 1 , 2 , positive alteration , respectively , of at least about 10 % , 25 % , 30 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 50 % , 75 % , or 100 % relative to a reference . 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , By " root mean square deviation ” is meant the square root 37 , 38 , 39 , 40 , 41, 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , or 50 . of the arithmetic mean of the squares of the deviations from Unless specifically stated or obvious from context, as the mean . used herein , the term “ or ” is understood to be inclusive . By " reducing cell survival” is meant to inhibit the viabil - 35 Unless specifically stated or obvious from context, as used ity of a cell or to induce cell death relative to a reference cell. herein , the terms “ a ” , “ an ” , and “ the” are understood to be By " reference ” is meant a standard or control condition . singular or plural. A “ reference sequence” is a defined sequence used as a The term “ including ” is used herein to mean , and is used basis for sequence comparison . A reference sequence may be interchangeably with , the phrase “ including but not limited a subset of or the entirety of a specified sequence ; for 40 to . " example , a segment of a full - length cDNA or gene sequence , Unless specifically stated or obvious from context, as or the complete cDNA or gene sequence . For polypeptides, used herein , the term “ about” is understood as within a range the length of the reference polypeptide sequence will gen of normal tolerance in the art, for example within 2 standard erally be at least about 16 amino acids , preferably at least deviations of the mean . About can be understood as within about 20 amino acids, more preferably at least about 25 45 10 % , 9 % , 8 % , 7 % , 6 % , 5 % , 4 % , 3 % , 2 % , 1 % , 0 . 5 % , 0 . 1 % , amino acids, and even more preferably about 35 amino 0 .05 % , or 0 .01 % of the stated value . Unless otherwise clear acids , about 50 amino acids, or about 100 amino acids . For from context, all numerical values provided herein are nucleic acids, the length of the reference nucleic acid modified by the term about. sequence will generally be at least about 50 nucleotides , The recitation of a listing of chemical groups in any preferably at least about 60 nucleotides, more preferably at 50 definition of a variable herein includes definitions of that least about 75 nucleotides , and even more preferably about variable as any single group or combination of listed groups . 100 nucleotides or about 300 nucleotides or any integer The recitation of an embodiment for a variable or aspect thereabout or therebetween . herein includes that embodiment as any single embodiment By “ subject ” is meant a mammal, including , but not or in combination with any other embodiments or portions limited to , a human or non -human mammal, such as a 55 thereof . bovine , equine , canine , ovine , or feline . Any compounds, compositions, or methods provided By “ specifically binds ” is meant a compound or antibody herein can be combined with one or more of any of the other that recognizes and binds a polypeptide of the invention , but compounds , compositions, and methods provided herein . which does not substantially recognize and bind other mol ecules in a sample , for example , a biological sample , which 60 DESCRIPTION OF THE DRAWINGS naturally includes a polypeptide of the invention . The term " sulfhydryl” or “ thiol” means — SH . FIG . 1 is a sequence comparison of human BRDT ( 1 ) with As used herein , the term “ tautomers” refers to isomers of human BRD4 ( 1) and mouse BRDT( 1) . Protein sequence organic molecules that readily interconvert by tautomeriza - alignment reveals a high degree of sequence identity tion , in which a hydrogen atom or proton migrates in the 65 between homologous and orthologous domains. Identical reaction , accompanied in sonic occasions by a switch of a ( red ) and similar (yellow ) residues are highlighted . Depicted single bond and an adjacent double bond . above the residue sequences are schematic representations US 9 , 789 ,120 B2 17 18 of major helical elements . Contacts between ( + ) - JQ1 and FIGS. 8A -8C show the effect of JQ1 on sperm count and BRDT ( 1 ) are depicted with a black star. The conserved testicular mass . In a repeat study , C57B6 mice were treated asparagine mediating acetyl- lysine recognition is depicted with JQ1 (50 mpkx8 weeks ) . FIG . 8A includes a graph with a blue star . showing sperm count in the test mice . FIG . 8B includes a FIGS. 2A -2C show BRDT inhibition by ( + ) - JQ1. FIG . 2A 5 graph showing testes weight in the test mice . FIG . 8C is the structure of the active ( + )- JQ1 enantiomer. FIG . 2B is includes phase contrast images of sperm from test mice . a plot showing the competitive inhibition of BRDT binding FIGS. 9A -9C show that the effects of JQ1 are reversed to synthetic biotinylated H4Kac4 by ( + ) - JQ1 ( IC50 : 11 nM ) upon removal. FIG . 9A includes a graph showing sperm using a proximity detection assay. FIG . 2C includes a graph motility levels in test mice twomonths and fourmonths after showing the results of the assay with 500 nM of the termination of JQ1 treatment. FIG . 9B includes a graph indicated compound . Error bars show standard deviation . showing testes weight in test mice two months and four FIG . 3 is a MAFFT alignment of human BRDT and months after termination of JQ1 treatment. FIG . 9C includes human BRD4 . a graph showing the sperm counts in test mice two months FIG . 4 is a MAFFT alignment of human BRDT and 15 and four months after termination of JQ1 treatment. mouse BRDT. FIGS . 5A -5H show gross and histological analysis of DETAILED DESCRIPTION OF THE testes from mice treated with JQ1 or vehicle control. FIG . INVENTION 5A includes an image providing a gross analysis of testes from 9 -meek old mice injected with control or JQ1. FIG . 5B 20 This invention is based , at least in part , on the discovery includes a graphical representation of testes weights (mg ) that a small -molecule inhibitor (JQ1 ) of the bromodomain from mice treated with control or JQ1 for 3 - 6 weeks, 6 - 9 and extra -terminal (BET ) subfamily of epigenetic reader weeks, or 6 - 12 weeks of age . Data represent the proteins is essential for chromatin remodeling during sper mean - standard error of the mean (SEM ) , and are annotated miogenesis . Biochemical analysis confirms that occupancy with P - values as obtained from a two - tailed t- test (* indi- 25 of the BRDT acetyl- lysine binding pocket by JQ1 prevents cates significant at P < 0 .05 ) . FIGS . 5C - 5F include histologi - recognition of acetylated histone H4 . The invention is also cal stains showing the histology of testes of 6 -week old mice based on the discovery that treatment of mice with JO1 treated with (FIG . 5C ) control or ( FIG . 5D ) JQ1 from 3 -6 reduced the number and motility of spermatozoa, as well as weeks of age , and 12 -week old mice treated with (FIG . 5E ) testis size . Although JQ1 -treated males mate normally , control or (FIG . 5F ) JQ1 from 6 - 12 weeks of age. Intertu - 30 inhibitory effects of JQ1 evident at the spermatocyte stage bular islands of Leydig cells are depicted with arrows in cause a dramatic decrease in fertilized oocytes and a revers FIGS . 5C -5F . vacuolization ( V ) is highlighted in ible contraceptive effect in males. Accordingly , the present several tubules in FIG . 5F . FIGS . 5G and 5H include invention is directed to a novel type of male contraceptive histological stains showing the histology of the epidid that can cross the blood : testis boundary and inhibits bro ymides from males treated with ( FIG . 5G ) control or ( FIG . 35 modomain activity during spermatogenesis . 5H ) JQ1 from 6 - 12 weeks of age . Fewer spermatozoa and Bromodomain - Containing Proteins multiple large nucleated cells ( black arrow ) are observed in Gene regulation is fundamentally governed by reversible , the epididymal lumen of the JQ1 - treated mice (FIG . 5H ) non - covalent assembly ofmacromolecules . Signal transduc compared to the control epididymal lumen (FIG . 5G ) , which tion to RNA polymerase requires higher- ordered protein is densely packed with mature spermatozoa . FIGS. 5C - 5H 40 complexes , spatially regulated by assembly factors capable were photographed at the same magnification . of interpreting the post - translational modification states of FIGS . 6A and 6B show epididymal sperm counts and chromatin . Epigenetic readers are structurally diverse pro fertilization potential. FIG . 6A is a graphical representation teins each possessing one or more evolutionarily conserved of the sperm counts obtained from the entire epididymides effector modules , which recognize covalent modifications of of males treated with JQ1 or control from 6 - 9 weeks of age 45 histone proteins or DNA . The e - N - acetylation of lysine or the tail ( cauda ) of the epididymis of males treated from residues (Kac ) on histone tails is associated with an open 6 - 12 weeks of age. FIG . 6B includes a graph showing the in chromatin architecture and transcriptional activation ". Con vitro developmental potential of oocytes obtained from text- specific , molecular recognition of acetyl- lysine is prin superovulated females mated to males treated for 5 weeks cipally mediated by bromodomains. with a control or JQ1. All data represent the mean SEM , and 50 Bromodomain -containing proteins are of substantial bio are annotated with P - values as obtained from a two - tailed logical interest, as components of transcription factor com t - test ( * indicates significant at P < 0 . 05 ) . plexes ( TAF1, PCAF, Gen5 and CBP ) and determinants of FIGS. 7A - 7C show the molecular analysis of the testes of epigenetic memory ' . There are 41 human proteins contain mice treated with JQ1 or control. FIG . 7A includes a graph ing a total of 57 diverse bromodomains . Despite large showing the quantitative RT- PCR results for males treated 55 sequence variations, all bromodomains share a conserved from 6 - 12 weeks of age with JQ1 or a control solution . The fold comprising a left -handed bundle of four alpha helices mouse genes tested were Plzf (promyelocytic leukemia (az , Q4, . B , ac) , linked by diverse loop regions (ZA and BC zinc - finger or Zbtb16 ) , Stra8 ( stimulated by retinoic acid loops ) that determine substrate specificity . Co - crystal struc gene 8 ) , Brdt (bromodomain , testis -specific ), Cenal (cyclin tures with peptidic substrates showed that the acetyl- lysine A1) , Histlhlt (histone cluster 1 , histone 1 , testis -specific ), 60 is recognized by a central hydrophobic cavity and is Papolb (poly ( A ) polymerase beta or Tpap ), Klf17 (Kruppel - anchored by a hydrogen bond with an asparagine residue like factor 17 or Zfp393 ), and Prml (protamine 1 ). Data present in most bromodomains" . The bromodomain and represent the mean : SEM , and are annotated with P - values extra- terminal (BET ) - family (BRD2 , BRD3, BRD4 and as obtained from a two - tailed t - test ( * indicates significant at BRDT ) shares a common domain architecture comprising P < 0 . 05 ; the P - value for Prm1 is 0 . 06 ) . FIGS . 7B and 7C 65 two N - terminal bromodomains that exhibit high level of include immunohistochemical staining images of TNP2 in sequence conservation , and a more divergent C - terminal control- treated (FIG . 7B ) and JQ1- treated ( FIG . 7C ) testes . recruitment domainº. US 9 ,789 , 120 B2 19 20 The invention features compositions and methods that are each Rz is independently selected from the group consist useful for inhibiting human bromodomain proteins . ing of: Compounds of the Invention ( i) H , aryl, substituted aryl, heteroaryl, or substituted The invention provides compounds ( e . g ., JQ1 and com heteroaryl; pounds of formulas delineated herein ) that bind in the 5 ( ii ) heterocycloalkyl or substituted heterocycloalkyl; binding pocket of the apo crystal structure of the first ( iii ) — C1 - C , alkyl, - C2- C , alkenyl or — C2- C , alky bromodomain of a BET family member ( e . g ., BRDT, BRD2, nyl, each containing 0 , 1 , 2 , or 3 heteroatoms BRD3 , BRD4 ). Without wishing to be bound by theory, selected from O , S , or N ; Cz- C12 cycloalkyl , these compounds are particularly effective in reducing male substituted — C3- C12 cycloalkyl, C3- C12 cycloalk fertility . In one approach , compounds useful for reducing 10 enyl, or substituted C3 -C12 cycloalkenyl, each of male fertility are selected using a molecular docking pro gram to identify compounds that are expected to bind to a which may be optionally substituted ; and bromodomain structural binding pocket . In certain embodi ( iv ) NH2, N = CR _ RO; ments , a compound of the invention can prevent, inhibit , or each R4 is independently H , alkyl, alkyl, cycloalkyl, disrupt , or reduce by at least 10 % , 25 % , 50 % , 75 % , or 100 % 15 heterocycloalkyl, aryl, or heteroaryl, each of which the biological activity of a BET family member ( e . g . , BRD2 , is optionally substituted ; BRD3 , BRD4, BRDT) and /or disrupt the subcellular local or Rz and R4 are taken together with the nitrogen atom ization of such proteins, e . g . , by binding to a binding site in to which they are attached to form a 4 - 10 -membered a bromodomain apo binding pocket. ring ; In certain embodiments , a compound of the invention is 20 Ro is alkyl, alkenyl, cycloalkyl, cycloalkenyl, hetero a small molecule having a molecular weight less than about cycloalkyl, aryl, or heteroaryl, each of which is 1000 daltons, less than 800 , less than 600 , less than 500 , less optionally substituted ; or R4 and R . are taken than 400 , or less than about 300 daltons. Examples of together with the carbon atom to which they are compounds of the invention include JQ1 and other com attached to form a 4 - 10 -membered ring ; pounds that bind the binding pocket of the apo crystal 25 m is 0 , 1 , 2 , or 3 ; structure of the first bromodomain of a BET family member provided that ( e .g ., BRD4 (hereafter referred to as BRD4 ( 1) ; PDB ID ( a ) if ring A is thienyl, X is N , R is phenyl or substituted 2OSS ). JQ1 is a novel thieno - triazolo - 1 ,4 -diazepine . The phenyl, R , is H , Rg is methyl, and R , is – (CH2 ) n - L , invention further provides pharmaceutically acceptable salts in which n is 1 and L is — CON ( R R ), then Rz of such compounds. 30 and R4 are not taken together with the nitrogen atom In one aspect, the compound is a compound of Formula to which they are attached to form a morpholino ring; (b ) if ring A is thienyl , X is N , R is substituted phenyl , R , is H , Rg is methyl, and Ry is ( CH2) n - L , in (I ) 35 which n is 1 and L is - CO - N (R3R4 ) , and one of Rz and R4 is H , then the other of Rz and R4 is not methyl , hydroxyethyl, alkoxy, phenyl , substituted phenyl, pyridyl or substituted pyridyl; and (RA ) m A ( c ) if ring A is thienyl, X is N , R is substituted phenyl, 40 R2 is H , Rg is methyl, and R , is (CH ) n- L , in which n is 1 and L is COOR3, then Rz is not methyl or ethyl ; or a salt , solvate or hydrate thereof. RB In certain embodiments , R is aryl or heteroaryl, each of 45 which is optionally substituted . wherein In certain embodiments , L is H , — C00Rz, - CON X is N or CR ; (R2R4 ) , - S ( O )2 - R3, - S ( O ) 2 - N (R3R4 ), N (R3R4 ) , N (R4 ) R , is H , alkyl, cycloalkyl, heterocycloalkyl, aryl, or C ( O ) Rz or optionally substituted aryl. In certain embodi heteroaryl, each of which is optionally substituted ; ments , each R , is independently selected from the group Rp is H , alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, 50 consisting of: H , C -C , alkyl , containing 0 , 1, 2 , or 3 haloalkyl, hydroxy , alkoxy, or — COO - R3, each of heteroatoms selected from O , S , or N ; or NH2, N = CR _Ro . which is optionally substituted ; In certain embodiments, R2 is H , D , halogen ormethyl . ring A is aryl or heteroaryl ; In certain embodiments , RB is alkyl, hydroxyalkyl, each R , is independently alkyl, cycloalkyl, heterocy - haloalkyl, or alkoxy ; each of which is optionally substituted . cloalkyl, aryl, or heteroaryl, each of which is option - 55 In certain embodiments , RB is methyl, ethyl, hydroxy ally substituted ; or any two R , together with the methyl, methoxymethyl, trifluoromethyl, COOH , COOMe, atoms to which each is attached , can form a fused COOEt, or COOCH OC (O )CHz . aryl or heteroaryl group ; In certain embodiments , ring A is a 5 or 6 -membered aryl R is alkyl , cycloalkyl, heterocycloalkyl, aryl, or het - or heteroaryl. In certain embodiments , ring A is thiofuranyl, eroaryl; each of which is optionally substituted ; 60 phenyl, naphthyl, biphenyl, tetrahydronaphthyl, indanyl , R is ( CH2) , -L , in which n is 0 - 3 and L is H , - C00 — pyridyl, furanyl, indolyl, pyrimidinyl, pyridizinyl, pyrazinyl , Rz, C0Rz, C0 — N RR( . ), - S ( O ) , Rz, imidazolyl , oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, - S ( O ) 2 - N (R3R4 ) , N (R3R4 ) , N (R4 ) C ( O ) Rz, option quinolinyl, pyrrolyl, pyrazolyl, or 5 ,6 , 7 , 8 -tetrahydroisoqui ally substituted aryl , or optionally substituted het - nolinyl. eroaryl; 65 In certain embodiments , ring A is phenyl or thienyl. R2 is H , D (deuterium ) , halogen , or optionally substituted In certain embodiments , m is 1 or 2 , and at least one alkyl; occurrence of R4 is methyl. US 9 ,789 , 120 B2 21 22 In certain embodiments , each R , is independently H , an In certain embodiments , Rg is methyl, ethyl, hydroxy optionally substituted alkyl , or any two R , together with the methyl, methoxymethyl, trifluoromethyl, COOH , COOMe, atoms to which each is attached, can form an aryl. COOEt, or COOCH OC ( O )CHz . In another aspect, the compound is a compound of For In certain embodiments , each R4 is independently an mula II: 5 optionally substituted alkyl, or any two R , together with the atoms to which each is attached , can form a fused aryl. In certain embodiments , each RA is methyl. ( II ) In another aspect, the compound is a compound of for mula III: 10 (RA ) / ( III)

RB (RA ) m + A wherein X is N or CR = ; 20 R , is H , alkyl, cycloalkyl, heterocycloalkyl, aryl, or RB heteroaryl, each of which is optionally substituted ; Rg is H , alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, wherein haloalkyl, hydroxy , alkoxy, or — COO R3, each of X is N or CR = ; which is optionally substituted ; R , is H , alkyl, cycloalkyl, heterocycloalkyl, aryl, or each RA is independently alkyl , cycloalkyl, heterocy heteroaryl, each of which is optionally substituted ; cloalkyl, aryl, orheteroaryl , each of which is optionally Rg is H , alkyl , hydroxylalkyl , aminoalkyl, alkoxyalkyl, substituted ; or any two R , together with the atoms to haloalkyl, hydroxy , alkoxy, or — C00 — R3, each of which each is attached , can form a fused aryl or which is optionally substituted ; heteroaryl group ; 30 ring A is aryl or heteroaryl; R is alkyl, cycloalkyl, heterocycloalkyl , aryl, or het each RA is independently alkyl, cycloalkyl, heterocy eroaryl, each of which is optionally substituted ; cloalkyl, aryl, or heteroaryl, each of which is option R ' , is H , COOR3, - CO R3, optionally substituted ally substituted ; or any two R , together with the aryl, or optionally substituted heteroaryl; 35 atoms to which each is attached , can form a fused each Rz is independently selected from the group con aryl or heteroaryl group ; sisting of: R is alkyl , cycloalkyl, heterocycloalkyl, aryl , or het ( i ) H , aryl, substituted aryl, heteroaryl, substituted eroaryl, each of which is optionally substituted ; heteroaryl; each Rz is independently selected from the group consist ( ii) heterocycloalkyl or substituted heterocycloalkyl; 40 ing of: ( iii ) C7 - C alkyl, C2- Cg alkenyl or C2- C , alky ( i ) H , aryl, substituted aryl, heteroaryl, or substituted nyl, each containing 0 , 1 , 2 , or 3 heteroatoms heteroaryl; selected from O , S , or N ; cycloalkyl, substituted ( ii ) heterocycloalkyl or substituted heterocycloalkyl; - Cz -C12 cycloalkyl, substituted Cz- C12 ( iii ) C1 - C , alkyl, - C2- C alkenyl or — C2- C alky cycloalkyl ; C3 -C12 cycloalkenyl, or substituted 45 nyl, each containing 0 , 1 , 2, or 3 heteroatoms - C3- C12 cycloalkenyl; each of which may be selected from O , S , or N ; C3- C12 cycloalkyl, optionally substituted ; substituted — C3- C12 cycloalkyl , - C3 - C12 cycloalk m is 0 , 1 , 2 , or 3 ; provided that if R ', is — COOR3, X enyl, or substituted C3- C12 cycloalkenyl, each of is N , R is substituted phenyl , and Rg is methyl , then Rz which may be optionally substituted ; and 50 ( iv ) NH2, N = CR _ RO; is not methyl or ethyl ; each R4 is independently H , alkyl, alkyl, cycloalkyl, or a salt, solvate or hydrate thereof. heterocycloalkyl, aryl, or heteroaryl, each of which In certain embodiments , R is aryl or heteroaryl, each of is optionally substituted ; which is optionally substituted . In certain embodiments , Ris or R , and R , are taken together with the nitrogen atom phenyl or pyridyl, each of which is optionally substituted. In 55 to which they are attached to form a 4 - 10 -membered certain embodiments , R is p - Cl- phenyl, o -Cl - phenyl , m - Cl ring ; phenyl, p - F -phenyl , o - F - phenyl, m - F -phenyl or pyridinyl. Ro is alkyl, alkenyl, cycloalkyl, cycloalkenyl, hetero In certain embodiments , R ' , is - C00 – Rz, optionally cycloalkyl, aryl, or heteroaryl, each of which is substituted aryl, or optionally substituted heteroaryl; and Rz optionally substituted ; or R . and R . are taken is C - C alkyl, which contains 0 , 1 , 2 , or 3 heteroatoms 60 together with the carbon atom to which they are selected from O , S , or N , and which may be optionally attached to form a 4 - 10 -membered ring ; substituted . In certain embodiments , R ', is — COORZ , and m is 0 , 1 , 2 , or 3 ; Rz is methyl, ethyl, propyl, i- propyl, butyl, sec -butyl , or provided that: t- butyl; or R ', is H or optionally substituted phenyl. ( a ) if ring A is thienyl, X is N , R is phenyl or substituted In certain embodiments , RB is methyl, ethyl, hydroxy 65 phenyl, RB is methyl, then R3 and R4 are not taken methyl, methoxymethyl, trifluoromethyl , COOH , COOMe, together with the nitrogen atom to which they are COOEt, COOCH OC ( O )CHz . attached to form a morpholino ring ; and US 9 ,789 , 120 B2 23 24 ( b ) if ring A is thienyl , X is N , R is substituted phenyl, ( iii) C7- C alkyl, C2 -C , alkenyl or - C2 - C , alky R , is H , RR is methyl, and one ofR2 and R , is H , then nyl, each containing 0 , 1 , 2 , or 3 heteroatoms the other of Rz and R4 is not methyl, hydroxyethyl, selected from O , S , or N ; Cz -C12 cycloalkyl, alkoxy, phenyl, substituted phenyl , pyridyl or sub substituted - C3 -C12 cycloalkyl, - C3- C12 cycloalk stituted pyridyl; and enyl, or substituted - Cz - C12 cycloalkenyl, each of or a salt , solvate or hydrate thereof. which may be optionally substituted ; and In certain embodiments , R is aryl or heteroaryl , each of (iv ) NH2, N = CRAR ; which is optionally substituted . In certain embodiments , Ris each R4 is independently H , alkyl, alkyl, cycloalkyl, phenyl or pyridyl , each of which is optionally substituted. heterocycloalkyl, aryl, or heteroaryl, each of which In certain embodiments , R is p - Cl- phenyl , o -Cl - phenyl , m - Cl- phenyl , p - F -phenyl , 0 - F -phenyl , m - F - phenyl or is optionally substituted ; pyridinyl. In certain embodiments , Rz is H , NH2, or or Rz and R4 are taken together with the nitrogen atom N = CR _ R .. to which they are attached to form a 4 - 10 -membered In certain embodiments , each Rd is independently H , 15 ring ; alkyl, cycloalkyl , heterocycloalkyl, aryl, heteroaryl; each of Ro is alkyl , alkenyl, cycloalkyl, cycloalkenyl, hetero which is optionally substituted . cycloalkyl, aryl , or heteroaryl, each of which is In certain embodiments , Ro is alkyl, alkenyl , cycloalkyl, optionally substituted ; or R4 and R . are taken heterocycloalkyl, aryl, or heteroaryl, each of which is together with the carbon atom to which they are optionally substituted . 20 attached to form a 4 - 10 -membered ring ; In another aspect, the compound is a compound of for m is 0 , 1 , 2 , or 3 ; mula IV : provided that ( a ) if ring A is thienyl, X is N , R , is H , Rg is methyl, (IV ) 25 and R , is (CH2 ) , - L , in which n is O and L is - CO - N ( R R _ ), then R , and R . are not taken together with the nitrogen atom to which they are attached to form a morpholino ring ; 30 ( b ) if ring A is thienyl , X is N , R2 is H , RB is methyl , and Ri is (CH ) n -L , in which n is O and L is - C0 — N (R2R4 ) , and one of R , and R4 is H , then the * R other of Rz and R4 is not methyl, hydroxyethyl, (RA ) m + A alkoxy, phenyl, substituted phenyl, pyridyl or sub . 35 stituted pyridyl; and ( c ) if ring A is thienyl, X is N , R , is H , Rg is methyl, di and R , is – (CH , ) , - L , in which n is 0 and L is RB COOR3, then R2 is not methyl or ethyl; or 40 a salt, solvate or hydrate thereof. wherein In certain embodiments , R , is (CH2 ), - L , in which n is X is N or CRS; 0 - 3 and L is COOR3, optionally substituted aryl, or optionally substituted heteroaryl ; and Rz is - C . - C , alkyl, Rs is H , allyl, cycloalkyl , heterocycloalkyl, aryl, or which contains 0 , 1 , 2 , or 3 heteroatoms selected from O , S , heteroaryl , each of which is optionally substituted ; 45a or N , and which may be optionally substituted . In certain Rg is H , alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, embodiments , n is 1 or 2 and L is alkyl or — COO - R3, and haloalkyl, hydroxy, alkoxy, or - C00 — Rz, each of Rz is methyl, ethyl, propyl, i -propyl , butyl, sec -butyl , or which is optionally substituted ; t - butyl; or n is 1 or 2 and L is H or optionally substituted ring A is aryl or heteroaryl; phenyl. each RA is independently alkyl, cycloalkyl, heterocy - > In certain embodiments , R2 is H or methyl. cloalkyl, aryl, or heteroaryl, each of which is option In certain embodiments , RB is methyl, ethyl, hydroxy ally substituted ; or any two R , together with the methyl, methoxymethyl, trifluoromethyl , COOH , COOMe, atoms to which each is attached , can form a fused COOE , COOCH _OC (O )CHz . aryl or heteroaryl group ; 55 In certain embodiments , ring A is phenyl, naphthyl, biphe R , is (CH2 ) n- L , in which n is 0 -3 and L is H , C00 nyl , tetrahydronaphthyl, indanyl, pyridyl, furanyl, indolyl, R3, C0R3, C0 — N (R3R4 ) , - S ( O ) 2 - R3, pyrimidinyl, pyridizinyl, pyrazinyl, imidazolyl, oxazolyl , S ( O 2) N (R3R . ), N (R3R4 ) , N ( R _ ) C ( O )Rz , option thienyl, thiazolyl, triazolyl, isoxazolyl , quinolinyl, pyrroly , ally substituted aryl, or optionally substituted het pyrazolyl , or 5 , 6 , 7 , 8 - tetrahydroisoquinolinyl. eroaryl ; In certain embodiments , each R , is independently an R2 is H , D , halogen , or optionally substituted alkyl; optionally substituted alkyl, or any two R , together with the each Rz is independently selected from the group consist atoms to which each is attached , can form an aryl. ing of: The methods of the invention also relate to compounds of ( i ) H , aryl, substituted aryl , heteroaryl, or substituted 65 Formulae V -XXII , and to any compound described herein . heteroaryl; In another aspect , the compound is a compound repre ( ii ) heterocycloalkyl or substituted heterocycloalkyl ; sented by the formula : US 9 , 789 , 120 B2 2525 26 - continued N

111lllll ??

NH

or a salt , solvate or hydrate thereof. In certain embodiments , the compound is ( + ) -JQ1 :

- Cl, or

or a salt , solvate or hydrate thereof. In another aspect, the compound is a compound repre - ze sented by the formula :

StringEN N OH ; NH2, N or a salt, solvate or hydrate thereof. In another aspect , the compound is a compound repre sented by the formula :

> - *1311

SN CO2H Spread or US 9 , 789 , 120 B2 28 - continued

NH

?llll

5N or a salt , solvate or hydrate thereof. -continued In another aspect , the compound is a compound repre JQIR sented by any one of the following formulae : 20 JQIS

N 0 ??25

??3 )

JQ6 35 JQ13

NH

N 0 ?4 { - NH NH ????.? 45 50 ? JQ11 ? JQ21 ? I ( 0 . ?55

?

: : TIIIII

66)

?65 US 9, 789 ,120 B2 29 30 -continued -continued J020 JQ18 ? *?

6

- KS1 J019

N0 ? i >

?

p ? or a salt, solvate or hydrate thereof. In another aspect , the compound is a compound repre JQ24B ? . Senited by ally One of the following forillulae :

B { OH }2 _ of

4f0

- NH 45

Q? B { OH )2

- NH N0 55 NH

?? or a salt, solvate or hydrate thereof. In another aspect, the compound is a compound repre sented by any one of the following structures: ?

US 9 , 789 ,120 B2 32 -continued

5

?? IIIlIlli

- NH

10 ? S0, Na

??IllIII 30 HN

NH

25

30

? ?? lIIIlli llllli NH NH2 35

4f0

; { H F ,

45

Illll ? HN?

50

55

N HN 60 - NH

NH

65 US 9 , 789 ,120 B2 33 34 -continued - continued

NH HN ?_ Fin:??. uik J ? \ ( PEG} 2 ? " NS ? 10

nullum 20

25

30 _

35 _

4f0

45

50 _

55

N HNHN 60 ??????65 US 9 , 789 , 120 B2 35 36 - continued -continued

•

•10

•

NH

2 ) NH |AI25

3 )

N EZ NHN •35

4 ) F F

EZ •45

•50)

Z

Z

•55

- NH ???? # N HNHN 60)

8

6

2 US 9 , 789 ,120 B2 37 38 -continued - continued >- F F # N HN

???-IIIIII NH

- or N HN >

> IIIIIII

NH ???? -

NHN > NH

or a salt, solvate or hydrate thereof . In certain embodiments , a compound of the invention can be represented by one of the following structures:

55

IIIIII , 2N HN > US 9 , 789 , 120 B2 39 40 - continued -continued

IIIIIIII IIIT

10

NH 10) - NH

25

3 )

HN EN HN 35

4 )

" lllllly 45 IIIIIIII # N HN. N2N HN -

50)

55

* IIIIII

HN EN HNH 60) ?????????? 6 US 9 , 789 ,120 B2 42 - continued - continued

itum{

} 10

N - N

or a salt , solvate or hydrate thereof. 20 In one embodiment , the compound is represented by the structure :

25

? ? ? IIIMI , ???& NH or a salt , solvate or hydrate thereof. In another embodiment, the compound is represented by the structure : $_

? ;_

- NH

;_

8_

20, _ or ? co _ or a salt, Solvate or lnydrate thereof. In another embodiment, the compound is represented by the structure : US 9 ,789 , 120 B2 44 43 -continued ( VI)

14Illl

N

RB ( VII )

or a salt, solvate or hydrate thereof. In certain embodiments , a compound of the invention can have the opposite chirality of any compound shown herein . 25 In certain embodiments , the compound is a compound represented by Formula ( V ) , (VI ) , or (VII ) : in which R , R1, and R , and RB have the same meaning as in ( V ) 30 Formula (I ) ; Y is O , N , S , or CRs, in which R , has the same meaning as in Formula ( I ) ; n is 0 or 1 ; and the dashed circle in Formula (VII ) indicates an aromatic or non - aromatic ring ; or a salt , solvate , or hydrate thereof. In certain embodiments of any of the Formulae I - IV and VI (or any formula herein ) , Ro represents the non - carbonyl portion of an aldehyde shown in Table A , below ( i. e . , for an aldehyde of formula RGCHO , R is the non - carbonyl portion of the aldehyde ) . In certain embodiments , R4 and R together represent the non - carbonyl portion of a ketone shown in Table A ( i. e ., for a ketone of formula RGC (OR4 , R , and R . are the non - carbonyl portion of the ketone ) . US 9 , 789 , 120 B2 45 46

HN O g'yxZI ZE Na+

o =S = o

-O

TABLEA Plate1 HO

Br N- 328972015-OH

HO HO

OH - US 9 , 789 , 120 B2 20 48

-Br

90

HO

TABLEA-continued OH HO. OHOH OH OH 0505 HOVY HO. HO -OH -OH

Br Basissage04 de .

C US 9 , 789, 120 B2 49 50

-O -OH Br

OH Br

TABLEA-continued Br OH HO —CI

H

Br HO

-OH

Br.

HO US 9 , 789 , 120 B2 51

HO

Br

OH TABLEA-continued Br HO

10 ??

$ 9. 95 F "acao US 9 , 789 , 120 B2 53 US9 , 789 ,120 B2 5454

o

HO TABLEA-continued HO HO

11 OH Ph Ph HON

-OH

HO OH HO) A 2 12 , D 2009E FO US 9 , 789 , 120 B2 55 56

03

TABLEA-continued Plate2Plate

02

01

A US 9 , 789 ,120 B2 57 58

? 8 H ? ?

TABLEA-continued NH

's

7

W

D US 9 , 789 , 120 B2 59 09

Br

1 8

É

LL(

TABLEA-continued CO

05

?? 04 ANH HN

? US 9 , 789 ,120 B2 z9_

60 Z - 26.

TABLEA-continued 8 HN _ 8 _08 _ 88

07

HO

F H B C US 9, 789 , 120 B2 | 6363 (644 ) Sss ?

TABLEA-continued ?HO

0 NH ? ??? US 9 , 789 , 120 B2 65 99

LL L O F 110 HO 1833 ES— O Z

HN TABLEA-continued HO =0 HO

O o ZE 10 IL 2 - 2

HO Br

A F US 9 , 789 ,120 B2 ? 67 89

N

F

TABLEA-continued Plate3

H0 | ?

G H US 9, 789 , 120 B2 6969 ? ? 70 ?

HC1

0|

~NH

0

? |

TABLEA-continued HO HO.

05

HO

LHH? 04

H US 9 , 789 ,120 B2

HN

TABLEA-continued

8 8? ???F US 9 , 789 , 120 B2 73 74

Z Z

TABLEA-continued O NH2 80

N

-

A o US 9 , 789 ,120 B2 75 ?

H }, 1/2H,0

0 fjH

TABLEA-continued

[ L

10

NH

?? _ o B US 9, 789 , 120 B2 77. 8

HN HO HOT

TABLEA-continued

D. G US 9, 789 , 120 B2 ? 6? 8008

? ? HN?

Plate4Plate4 TABLEA-continued OH

02 0

OH ?

FF

F ? NIN US 9 , 789 , 120 B2

OH

L L 11110 TJ 1

IMIT F

lll | HO 1

L L

TABLEA-continued

OH -

: 04

A US 9 , 789, 120 B2 8 04

OH O ?

HT TABLEA-continued 0= HO.

F 08 F

F ,H2N

NH H=C HC0

H—CI 07

? OH

A

E FF A US 9 , 789 , 120 B2 85 98

NH2

O OH

TABLEA-continued NTYao

OH 1 T

NS

. ? US 9 , 789 ,120 B2 87 88

HO.

0

NCH LL ??? r

TABLEA-continued =0 ??? - %0=

?4 ? ?? US 9 , 789 , 120 B2 89 90

TABLEA-continued

H US 9, 789 ,120 B2 91 _ 92 In one embodiment, the compound is a compound is - continued represented by the formula : ( VIII )

?

5

- RYR or a salt , Solvate , ur lnydrate thereof. _ n = 1 , 2 , 3 In certainermib0diments , the Comp0undis racemlic ) JOl ; ( XI} in certainleimb0dirmlents, the c0mp0undis ( * )- JOl . Incertain embodiments , the compound is a compound selected from 20 the group consisting of:

. Cl 25

NRR n = 1 , 2 , 3 30 _ NH , and ( XII ) ( 4 ) 35

4f0

NH

N ' 45 R ` 0H, n = 1 , 2, 3 of a salt , Solvate , or hydrate thereof. 50 Additional examples of compounds include compounds ( XIII} according to any of the follow formulae : (IX )

55 ?????

0 _ n = 1 , 2 , 3 n = 1 , 2 , 3 R - H , D , Me US 9 ,789 , 120 B2 93 94 - continued -continued (XIV ) ( XIX )

Ph

te?

ato R ' = H , D ,Me n = 1 , 2 , 3 (XV )

R "

N - - Me ?

R ' = OME, CH2OH , CH2NH2, CH2OMe (XXI )

????? Ž

to (XVII ) (XXII )

AN

(XVIII ) RR het 55 besta substitutedIn Formulae aryl , IX heteroaryl - XXII, R , andheteroaryl R ' can, heterocycloalkylbe, e .g ., H , aryl, C , - C , alkyl, - C , - C , alkenyl, - C2 - C , alkynyl, - C3 -C12 cycloalkyl, substituted C2- C12 cycloalkyl, C3- C12 cycloalkenyl, or substituted Cz- C2 cycloalkenyl , each of on which may be optionally substituted . In Formulae XIV , X can be any substituent for an aryl group as described herein . Compounds of the invention can be prepared by a variety of methods , some of which are known in the art. For instance, the chemical Examples provided hereinbelow pro 65 vide synthetic schemes for the preparation of the compound Also 2 - and 4 JO1 ( as the racemate ) and the enantiomers ( + ) - JQ1 and (- ) - JQ1 ( see Schemes S1 and S2 ). A variety of compounds US 9 , 789, 120 B2 95 96 of Formulae ( ID - ( VIII ) can be prepared by analogous meth For example , starting from JQ1, the analogous amine can ods with substitution of appropriate starting materials . be prepared as shown in Scheme 1 , below .

Scheme 1

1 ) DPPA, NEty - C HCOOH TN – 2 ) BzOH

NHCbz OH BBf3 JQ1 -- -????

NaH , Mel 1 ) RCHO SN < CI - { } C1C “ )RCEO - N ) N N 2 ) NaBH ( OAc) ' - - - ?- -- -

As shown in Scheme 1 , hydrolysis of the t - butyl ester of JQ1 affords the carboxylic acid , which is treated with diphenylphosphoryl azide (DPPA ) and subjected to Curtius 45 rearrangement conditions to provide the CbZ - protected amine , which is then deprotected to yield the amine . Sub sequent elaboration of the amine group , e. g ., by reductive amination yields secondary amines, which can be further alkylated to provide tertiary amines.

Scheme 2

HN - F ??? US 9 ,789 , 120 B2 97 US9,780 , 12082 98 - continued HNMoto HNsa 80

Scheme 2 shows the synthesis of further examples of the cores and /or aryl group appendages ( corresponding to group compounds of the invention , e. g ., of Formula I, in which the Rin Formula 1 ). Such aminodiarylketones are commercially fused ring core is modified ( e . g . , by substitution of a 30 available or can be prepared by a variety of methods , some different aromatic ring as Ring A in Formula I ). Use of aminodiarylketones having appropriate functionality ( e . g ., of which are known in the art. in place of the aminodiarylketone S2 in Scheme Si, infra ) Scheme 3 provides additional exemplary synthetic provides new compounds having a variety of fused ring schemes for preparing further compounds of the invention .

Scheme 3

DAM DAM HN 1 ) Base - CI LDA , DAMBI 2 ) D20 , or 07 Mel 03

fotoreAcid

HN poz US 9 , 789 , 120 B2 99 100 As shown in Scheme 3 , a fused bicyclic precursor ( see -continued Scheme S1, infra , for synthesis of this compound ) is func tionalized with a moiety R (DAM = dimethylaminomethylene protecting group ) and then elaborated by reaction with a to form the 5 tricyclic fused core . Substituent Rr can be varied by selec tion of a suitable hydrazide. N ' Additional examples of compounds of the invention (which can be prepared by the methods described herein ) 10 include : Amides: Amides can be prepared , e. g. , by preparation of a corre sponding carboxylic acid or ester , followed by amidation with an appropriate amine using standard conditions . In 15 certain embodiments , an amide provides a two -carbon " linker ” with a terminal nitrogen - containing ring ( e . g . , pyridyl, piperidyl , piperazinyl, imidazolyl (including N -methyl - imidazolyl ) , morpholinyl, and the like . Exem - 20 plary amide structures include :

25 NH

?? da

$ NH

# The use of a two -carbon linker between the amide moiety and the terminal nitrogen - containing ring is preferred .

NH “ Reverse Amides " : US 9 , 789 , 120 B2 101 102 - continued - continued

- CI

?

IZ

E HN

sition can be different

?

?k

30 N NH N

35 Secondary Amines :

NH NH

-Z - C1 HO N US 9 , 789 , 120 B2 103 104 - continued -continued B ( OH )2

10 N DZ

15 In certain embodiments , a compound having at least one chiral center is present in racemic form . In certain embodi ments, a compound having at least one chiral center is enantiomerically enriched , i . e . , has an enantiomeric excess 20 ( e . e . ) of at least about 10 % , 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 85 % , 90 % , 90 % , 95 % , 99 % , 99 % or 100 % . In certain embodiments , a compound has the same absolute configuration as the compound ( + ) - JQ1 ( ( S ) - tert - Butyl 2 - ( 4 ( 4 - chlorophenyl) - 2 , 3 , 9 -trimethyl -6H - thieno [ 3 , 2 - f ] [ 1 , 2 , 4 ] tri 25 azolo [ 4 , 3 - a ] [ 1 , 4 ]diazepin -6 - yl) acetate ) described herein . In certain embodiments of any of the Formulae disclosed herein , the compound is not represented by the following structure : 30 HzC

R ' , RA

- C1 $

# in which : HN R ', is C , - C4 alkyl; R ' 2 is hydrogen , halogen , or CZ -C4 alkyl optionally sub stituted with a halogen atom or a hydroxyl group ; 50 R 'z is a halogen atom , phenyl optionally substituted by a Boronic Acids: halogen atom , C , -C4 alkyl , CZ -C4 alkoxy, or cyano ; - NR , (CH2 ) m R6 wherein R , is a hydrogen atom or C1 -C4 alkyl , m is an integer of 0 - 4 , and Ro is phenyl or pyridyl optionally substituted by a halogen atom ; or B (OH )2 55 — NR C0 — (CH2 ) n - R , wherein R , is a hydrogen atom or C , -C4 alkyl, n is an integer of 0 - 2 , and Rg is phenyl or pyridyl optionally substituted by a halogen atom ; and R ' 4 is ( CH2) CONH R , wherein a is an integer of 1 - 4 , and R , is C1- C4 alkyl; C7 -C4 hydroxyalkyl; C . - C4 60 alkoxy ; or phenyl or pyridyl optionally substituted by C - C4 alkyl, C . - C4 alkoxy, amino or a hydroxyl group or NH (CH2 ) 6 COOR10 wherein b is an integer of 1 - 4 , and R10 is C , - C4 alkyl. The term “ pharmaceutically acceptable salt ” also refers to SN 65 a salt prepared from a compound disclosed herein (e . g ., JQ1, a compound of Formulas 1 - XXII) or any other compound delineated herein , having an acidic functional group , such as US 9 ,789 , 120 B2 105 106 a carboxylic acid functional group, and a pharmaceutically In embodiments , the inhibitor is used in combination with acceptable inorganic , or organic base . Suitable bases at least one additional male contraceptive agent or device . In include, but are not limited to , hydroxides of alkali metals related embodiments, the additionalmale contraceptive is a such as sodium , potassium , and lithium ; hydroxides of condom . In other related embodiments , the additional male alkaline earth metal such as calcium and magnesium ; 5 contraceptive is a modulator of testosterone production , hydroxides of other metals , such as aluminum and zinc ; androgen receptor function or stability . ammonia, and organic amines, such as unsubstituted or Pharmaceutical Compositions hydroxy - substituted mono -, di- , or trialkylamines ; dicyclo The invention features pharmaceutical compositions that contain one or more of the compounds described herein , a hexylamine ; tributyl amine ; pyridine; N -methyl , N - ethylam 10 derivative thereof, or a pharmaceutically acceptable salt or ine ; diethylamine; triethylamine ; mono -, bis - , or tris - ( 2 prodrug thereof as the active ingredient( s ). The pharmaceu hydroxy - lower alkyl amines ), such as mono -, bis -, or tris tical compositions contain a pharmaceutically acceptable ( 2 -hydroxyethyl ) -amine , 2 -hydroxy - tert- butylamine , or tris carrier , excipient, or diluent, which includes any pharma (hydroxymethyl ) methylamine , N , N ,- di - lower alkyl- N ceutical agent that does not itself induce the production of an (hydroxy lower alkyl) - amines , such as N , N - dimethyl- N - ( 2 15 immune response harmful to a subject receiving the com hydroxyethyl) - amine, or tri - ( 2 -hydroxyethyl ) amine ; position , and which may be administered without undue N -methyl - D - glucamine; and amino acids such as arginine , toxicity . As used herein , the term “ pharmaceutically accept lysine , and the like . The term “ pharmaceutically acceptable able ” means being approved by a regulatory agency of the salt ” also refers to a salt prepared from a compound dis - Federal or a state government or listed in the U . S . Pharma closed herein , or any other compound delineated herein , 20 copeia , European Pharmacopeia or other generally recog having a basic functional group , such as an amino functional nized pharmacopeia for use in mammals , and more particu group , and a pharmaceutically acceptable inorganic or larly in humans. These compositions can be useful as a male organic acid . Suitable acids include , but are not limited to , contraceptive. hydrogen sulfate , citric acid , acetic acid , oxalic acid , hydro A thorough discussion of pharmaceutically acceptable chloric acid , hydrogen bromide , hydrogen iodide , nitric acid , 25 carriers, diluents , and other excipients is presented in Rem phosphoric acid , isonicotinic acid , lactic acid , salicylic acid , ington ' s Pharmaceutical Sciences ( 17th ed ., Mack Publish tartaric acid , ascorbic acid , succinic acid , maleic acid , ing Company ) and Remington : The Science and Practice of besylic acid , fumaric acid , glucosic acid , glucaronic acid , Pharmacy (21st ed . , Lippincott Williams & Wilkins) , which saccharic acid , formic acid , benzoic acid , glutamic acid , are hereby incorporated by reference . The formulation of the methanesulfonic acid , ethanesulfonic acid , benzenesulfonic 30 pharmaceutical composition should suit themode of admin acid , and p - toluenesulfonic acid . istration . In embodiments , the pharmaceutical composition Methods of the Invention is suitable for administration to humans, and can be sterile , The present invention also relates to using the novel non -particulate and / or non - pyrogenic . compounds described herein , as well as other inhibitors of Pharmaceutically acceptable carriers , excipients , or BRDT as male contraceptives . Such compounds are known 35 diluents include , but are not limited , to saline , buffered in the art and described , for example , in WO2009084693 or saline, dextrose , water, glycerol, ethanol, sterile isotonic corresponding US2010286127 . aqueous buffer, and combinations thereof. Thus, in one aspect, the invention provides methods for Wetting agents, emulsifiers and lubricants , such as sodium reducing or inhibiting spermatozoa emission involving lauryl sulfate and magnesium stearate , as well as coloring administering an effective amount of a BRDT inhibitor to a 40 agents , release agents , coating agents , sweetening , flavoring male subject. In embodiments , the inhibitor is a compound and perfuming agents , preservatives, and antioxidants can having a formula delineated herein , a derivative thereof, or also be present in the compositions. a pharmaceutically acceptable salt or prodrug thereof. Examples of pharmaceutically -acceptable antioxidants In embodiments , the methods involve administering the include , but are not limited to : ( 1 ) water soluble antioxi inhibitor in an amount sufficient to suppress spermatogen - 45 dants , such as ascorbic acid , cysteine hydrochloride , sodium esis . bisulfate , sodium metabisulfite , sodium sulfite and the like ; In embodiments , the methods involve administering the (2 ) oil - soluble antioxidants , such as ascorbyl palmitate , inhibitor in an amount sufficient to induce azoospermia or butylated hydroxyanisole (BHA ) , butylated hydroxytoluene oligozoospermia . (BHT ) , lecithin , propyl gallate , alpha - tocopherol, and the In embodiments , the methods involve administering the 50 like ; and ( 3 ) metal chelating agents, such as citric acid , inhibitor in an amount sufficient to lower the spermatozoa ethylenediamine tetraacetic acid (EDTA ) , sorbitol, tartaric concentration to not more than 3 million /mL , 2 million /mL , acid , phosphoric acid , and the like . 1 million /mL , 0 . 5 million /mL , 0 . 25 million /mL , or 0 . 1 In embodiments , the pharmaceutical composition is pro million /mL . In related embodiments , the methods involve vided in a solid form , such as a lyophilized powder suitable administering the inhibitor in an amount sufficient to lower 55 for reconstitution , a liquid solution , suspension , emulsion , the spermatozoa concentration to not more than 0 . 1 million / tablet , pill, capsule , sustained release formulation , or pow mL . der . In embodiments , the inhibitor is administered in combi In embodiments , the pharmaceutical composition is sup nation with a pharmaceutically acceptable carrier, excipient, plied in liquid form , for example , in a sealed container or diluent . 60 indicating the quantity and concentration of the active In embodiments , the inhibitor is administered to the ingredient in the pharmaceutical composition . In related subject orally , transdermally , or by injection . In related embodiments , the liquid form of the pharmaceutical com embodiments , the inhibitor is administered in the form of a position is supplied in a hermetically sealed container . tablet or capsule . In related embodiments , the inhibitor is Methods for formulating the pharmaceutical composi administered by parenteral injection , intramuscular injec - 65 tions of the present invention are conventional and well tion , intravenous injection , subcutaneous implantation , sub - known in the art (see Remington and Remington ' s ) . One of cutaneous injection , or transdermal preparation . skill in the art can readily formulate a pharmaceutical US 9 , 789 , 120 B2 107 108 composition having the desired characteristics ( e . g . , route of or hydroxypropylmethyl cellulose ), lubricants , inert administration , biosafety , and release profile ) . diluents , preservatives , disintegrants ( for example , sodium Methods for preparing the pharmaceutical compositions starch glycolate or cross - linked sodium carboxymethyl cel include the step of bringing into association the active lulose ) , surface - actives , and /or dispersing agents . Molded ingredient with a pharmaceutically acceptable carrier and , 5 tablets can be made by molding in a suitable machine a optionally , one or more accessory ingredients . The pharma - mixture of the powdered active ingredient moistened with an ceutical compositions can be prepared by uniformly and inert liquid diluent. intimately bringing into association the active ingredient The tablets and other solid dosage forms, such as dragees , with liquid carriers , or finely divided solid carriers , or both , capsules , pills, and granules , can optionally be scored or and then , if necessary , shaping the product. Additional 10 prepared with coatings and shells , such as enteric coatings methodology for preparing the pharmaceutical composi- and other coatings well -known in the art. tions, including the preparation of multilayer dosage forms, The pharmaceutical compositions can also be formulated are described in Ansel' s Pharmaceutical Dosage Forms and so as to provide slow , extended , or controlled release of the Drug Delivery Systems ( 9th ed ., Lippincott Williams & active ingredient therein using, for example , hydroxypropy Wilkins ) , which is hereby incorporated by reference . 15 lmethyl cellulose in varying proportions to provide the Methods of Delivery desired release profile , other polymer matrices , liposomes The pharmaceutical compositions of the present invention and /or microspheres. The pharmaceutical compositions can can be administered to a subject by oral and non -oral means also optionally contain opacifying agents and may be of a ( e . g . , topically , transdermally, or by injection ). Such modes composition that releases the active ingredient( s ) only , or of administration and the methods for preparing an appro - 20 preferentially, in a certain portion of the gastrointestinal priate pharmaceutical composition for use therein are tract, optionally , in a delayed manner. Examples of embed described in Gibaldi' s Drug Delivery Systems in Pharma- ding compositions include polymeric substances and waxes . ceutical Care (1st ed ., American Society of Health -System The active ingredient can also be in micro - encapsulated Pharmacists ) , which is hereby incorporated by reference. form , if appropriate , with one or more pharmaceutically In embodiments , the pharmaceutical compositions are 25 acceptable carriers , excipients , or diluents well - known in the administered orally in a solid form . art ( see , e . g . , Remington and Remington ' s ) . Pharmaceutical compositions suitable for oral adminis - The pharmaceutical compositions can be sterilized by , for tration can be in the form of capsules, cachets , pills , tablets , example , filtration through a bacteria -retaining filter, or by lozenges ( using a flavored basis , usually sucrose and acacia incorporating sterilizing agents in the form of sterile solid or tragacanth ), powders , granules , or as a solution or a 30 compositions which can be dissolved in sterile water , or suspension in an aqueous or non -aqueous liquid , or as an some other sterile injectable medium immediately before oil - in -water or water - in - oil liquid emulsion , or as an elixir or use . syrup , or as pastilles ( using an inert base , such as gelatin and In embodiments , the pharmaceutical compositions are glycerin , or sucrose and acacia ) and /or as mouth washes and administered orally in a liquid form . the like , each containing a predetermined amount of a 35 Liquid dosage forms for oral administration of an active compound ( s ) described herein , a derivative thereof, or a ingredient include pharmaceutically acceptable emulsions, pharmaceutically acceptable salt or prodrug thereof as the microemulsions, solutions , suspensions, syrups and elixirs. active ingredient ( s ) . The active ingredient can also be In addition to the active ingredient, the liquid dosage forms administered as a bolus , electuary , or paste. can contain inert diluents commonly used in the art , such as, In solid dosage forms for oral administration ( e . g ., cap - 40 for example , water or other solvents , solubilizing agents and sules, tablets , pills , dragees, powders , granules and the like ), emulsifiers , such as ethyl alcohol, isopropyl alcohol, ethyl the active ingredient is mixed with one or more pharmaceu - carbonate, ethyl acetate , benzyl alcohol, benzyl benzoate , tically acceptable carriers , excipients , or diluents, such as propylene glycol, 1 , 3 - butylene glycol, oils ( in particular, sodium citrate or dicalcium phosphate, and / or any of the cottonseed , groundnut , corn , germ , olive, castor and sesame following: ( 1 ) fillers or extenders , such as starches, lactose , 45 oils ) , glycerol, tetrahydrofuryl alcohol, polyethylene glycols sucrose , glucose , mannitol, and /or silicic acid ; ( 2 ) binders , and fatty acid esters of sorbitan , and mixtures thereof. In such as , for example , carboxymethylcellulose , alginates , addition to inert diluents , the liquid pharmaceutical compo gelatin , polyvinyl pyrrolidone , sucrose and / or acacia ; ( 3 ) sitions can include adjuvants such as wetting agents ; emul humectants , such as glycerol; ( 4 ) disintegrating agents , such sifying and suspending agents , sweetening , flavoring , col as agar - agar, calcium carbonate , potato or tapioca starch , 50 oring , perfuming and preservative agents , and the like . alginic acid , certain silicates , and sodium carbonate ; ( 5 ) Suspensions , in addition to the active ingredient( s ) can solution retarding agents , such as paraffin ; ( 6 ) absorption contain suspending agents such as , but not limited to , accelerators , such as quaternary ammonium compounds ; ( 7 ) ethoxylated isostearyl alcohols , polyoxyethylene sorbitol wetting agents , such as , for example , acetyl alcohol and and sorbitan esters, microcrystalline cellulose , aluminum glycerol monostearate ; ( 8 ) absorbents, such as kaolin and 55 metahydroxide , bentonite , agar - agar and tragacanth , and bentonite clay ; ( 9 ) lubricants , such a talc , calcium stearate , mixtures thereof. magnesium stearate , solid polyethylene glycols , sodium In embodiments , the pharmaceutical compositions are lauryl sulfate , and mixtures thereof; and ( 10 ) coloring administered by non -oral means such as by topical applica agents . In the case of capsules, tablets , and pills , the phar- tion , transdermal application , injection , and the like . In maceutical compositions can also comprise buffering agents . 60 related embodiments , the pharmaceutical compositions are Solid compositions of a similar type can also be prepared administered parenterally by injection , infusion , or implan using fillers in soft and hard - filled gelatin capsules, and tation ( e .g . , intravenous, intramuscular, intraarticular, sub excipients such as lactose or milk sugars , as well as high cutaneous , and the like ) . molecular weight polyethylene glycols and the like. Compositions for parenteral use can be presented in unit A tablet can be made by compression or molding, option - 65 dosage forms, e. g . in ampoules or in vials containing several ally with one or more accessory ingredients . Compressed doses , and in which a suitable preservative can be added . tablets can be prepared using binders ( for example , gelatin Such compositions can be in form of a solution , a suspen US 9 , 789 ,120 B2 109 110 sion , an emulsion , an infusion device , a delivery device for Biocompatible carriers which can be used when formu implantation , or it can be presented as a dry powder to be lating a controlled release parenteral formulation include reconstituted with water or another suitable vehicle before carbohydrates such as dextrans, proteins such as albumin , use . One or more co - vehicles, such as ethanol, can also be lipoproteins or antibodies . employed . Apart from the active ingredient( s ) , the compo - 5 Materials for use in implants can be non -biodegradable , sitions can contain suitable parenterally acceptable carriers e . g ., polydimethylsiloxane , or biodegradable such as, e . g . , and / or excipients or the active ingredient ( s ) can be incor porated into microspheres , microcapsules , nanoparticles , poly (caprolactone ), poly (lactic acid ), poly ( glycolic acid ) or liposomes , or the like for controlled release . Furthermore , poly (ortho esters ). the compositions can also contain suspending, solubilising 10 h In embodiments , the active ingredient( s ) are administered stabilising , pH - adjusting agents, and / or dispersing agents . by aerosol. This is accomplished by preparing an aqueous The pharmaceutical compositions can be in the form of aerosol, liposomal preparation , or solid particles containing sterile injections. To prepare such a composition , the active the compound . A nonaqueous ( e . g ., fluorocarbon propellant) ingredient is dissolved or suspended in a parenterally accept suspension can be used . The pharmaceutical composition able liquid vehicle . Exemplary vehicles and solvents is15 cancan also be administered using a sonic nebulizer, which include , but are not limited to , water, water adjusted to a would minimize exposing the agent to shear, which can suitable by addition of an appropriate amount of hydrochlo - result in degradation of the compound . ric acid , sodium hydroxide or a suitable buffer, 1, 3 -butane Ordinarily , an aqueous aerosol is made by formulating an diol, Ringer 's solution and isotonic sodium chloride solu aqueous solution or suspension of the active ingredient ( s ) tion . The pharmaceutical composition can also contain one 20 together with conventional pharmaceutically -acceptable car or more preservatives, for example , methyl, ethyl or n - pro riers and stabilizers . The carriers and stabilizers vary with pyl p -hydroxybenzoate . To improve solubility , a dissolution the requirements of the particular compound , but typically enhancing or solubilising agent can be added or the solvent include nonionic surfactants ( Tweens , Pluronics, or polyeth can contain 10 -60 % w / w of propylene glycol or the like . ylene glycol) , innocuous proteins like serum albumin , sor The pharmaceutical compositions can contain one or 25 bitan esters , oleic acid , lecithin , amino acids such as glycine , more pharmaceutically acceptable sterile isotonic aqueous buffers, salts , sugars or sugar alcohols . Aerosols generally or nonaqueous solutions , dispersions, suspensions or emul- are prepared from isotonic solutions . sions , or sterile powders, which can be reconstituted into Dosage forms for topical or transdermal administration of sterile injectable solutions or dispersions just prior to use . an active ingredient ( s ) includes powders , sprays, ointments , Such pharmaceutical compositions can contain antioxidants ; 30 pastes, creams, lotions, gels , solutions , patches and inhal buffers; bacteriostats ; solutes , which render the formulation ants . The active ingredient( s ) can be mixed under sterile isotonic with the blood of the intended recipient; suspending conditions with a pharmaceutically acceptable carrier , and agents ; thickening agents ; preservatives ; and the like. with any preservatives, buffers , or propellants as appropri Examples of suitable aqueous and nonaqueous carriers , ate . which can be employed in the pharmaceutical compositions 35 Transdermal patches suitable for use in the present inven of the invention include water , ethanol, polyols ( such as tion are disclosed in Transdermal Drug Delivery : Develop glycerol, propylene glycol, polyethylene glycol, and the mental Issues and Research Initiatives (Marcel Dekker Inc ., like ) , and suitable mixtures thereof, vegetable oils , such as 1989 ) and U . S . Pat . Nos . 4 ,743 ,249 , 4 , 906 ,169 , 5 , 198 ,223 , olive oil , and injectable organic esters , such as ethyl oleate . 4 ,816 , 540 , 5 ,422 , 119 , 5 , 023 , 084 , which are hereby incorpo Proper fluidity can be maintained , for example , by the use of 40 rated by reference. The transdermal patch can also be any coating materials , such as lecithin , by the maintenance of the transdermal patch well - known in the art, including transs required particle size in the case of dispersions, and by the crotal patches . Pharmaceutical compositions in such trans use of surfactants . dermal patches can contain one or more absorption enhanc In some embodiments , in order to prolong the effect of an ers or skin permeation enhancers well- known in the art ( see , active ingredient, it is desirable to slow the absorption of the 45 e .g . , U .S . Pat. Nos. 4 ,379 , 454 and 4 , 973 ,468 , which are compound from subcutaneous or intramuscular injection . hereby incorporated by reference ). Transdermal therapeutic This can be accomplished by the use of a liquid suspension systems for use in the present invention can be based on of crystalline or amorphous material having poor water iontophoresis , diffusion , or a combination of these two solubility . The rate of absorption of the active ingredient effects . then depends upon its rate of dissolution which , in turn , can 50 Transdermal patches have the added advantage of pro depend upon crystal size and crystalline form . Alternatively, viding controlled delivery of active ingredient( s ) to the body . delayed absorption of a parenterally - administered active Such dosage forms can be made by dissolving or dispersing ingredient is accomplished by dissolving or suspending the the active ingredient ( s ) in a proper medium . Absorption compound in an oil vehicle . In addition , prolonged absorp - enhancers can also be used to increase the flux of the active tion of the injectable pharmaceutical form can be brought 55 ingredient across the skin . The rate of such flux can be about by the inclusion of agents that delay absorption such controlled by either providing a rate controlling membrane as aluminum monostearate and gelatin . or dispersing the active ingredient( s ) in a polymer matrix or Controlled release parenteral compositions can be in form gel . of aqueous suspensions, microspheres , microcapsules , mag - Such pharmaceutical compositions can be in the form of netic microspheres , oil solutions , oil suspensions , emul- 60 creams, ointments , lotions , liniments , gels , hydrogels , solu sions , or the active ingredient can be incorporated in bio - tions, suspensions , sticks , sprays , pastes , plasters and other compatible carrier( s ) , liposomes, nanoparticles , implants or kinds of transdermal drug delivery systems. The composi infusion devices. tions can also include pharmaceutically acceptable carriers Materials for use in the preparation of microspheres or excipients such as emulsifying agents , antioxidants , buff and / or microcapsules include biodegradable/ bioerodible 65 ering agents , preservatives , humectants , penetration enhanc polymers such as polyglactin , poly -( isobutyl cyanoacrylate ), ers , chelating agents , gel- forming agents , ointment bases, poly ( 2 - hydroxyethyl - L - glutamine ) and poly ( lactic acid ) . perfumes, and skin protective agents . US 9 , 789 ,120 B2 111 112 Examples of emulsifying agents include, but are not other silicone rubbers . Matrix materials can be insoluble limited to , naturally occurring gums, e . g . gum acacia or gum polypropylene , polyethylene, polyvinyl chloride, ethylvinyl tragacanth , naturally occurring phosphatides , e . g . soybean acetate , polystyrene and polymethacrylate , well as glycerol lecithin and sorbitan monooleate derivatives . esters of the glycerol palmitostearate , glycerol stearate , and Examples of antioxidants include, but are not limited to , 5 glycerol behenate type . Materials can be hydrophobic or butylated hydroxy anisole (BHA ) , ascorbic acid and deriva - hydrophilic polymers and optionally contain solubilising tives thereof, tocopherol and derivatives thereof, and cyste - agents . ine . Subcutaneous implant devices can be slow - release cap Examples of preservatives include , but are not limited to , sules made with any suitable polymer, e .g . , as described in parabens, such as methyl or propyl p -hydroxybenzoate and 10 U . S . Pat. Nos. 5 ,035 ,891 and 4 , 210 ,644 , which are hereby benzalkonium chloride . incorporated by reference . Examples of humectants include , but are not limited to , In general, at least four different approaches are appli glycerin , propylene glycol, sorbitol and urea . cable in order to provide rate control over the release and Examples of penetration enhancers include , but are not transdermal permeation of a drug compound . These limited to , propylene glycol, DMSO , triethanolamine , N , N - 15 approaches are : membrane -moderated systems, adhesive dimethylacetamide , N , N -dimethylformamide , 2 -pyrrolidone diffusion - controlled systems, matrix dispersion -type sys and derivatives thereof, tetrahydrofurfuryl alcohol, propyl- tems and microreservoir systems. It is appreciated that a ene glycol, diethylene glycol monoethyl or monomethyl controlled release percutaneous and /or topical composition ether with propylene glycol monolaurate or methyl laurate , can be obtained by using a suitable mixture of these eucalyptol, lecithin , Transcutol® , and Azone® . 20 approaches . Examples of chelating agents include , but are not limited In a membrane -moderated system , the active ingredient is to , sodium EDTA , citric acid and phosphoric acid . present in a reservoir which is totally encapsulated in a Examples of gel forming agents include , but are not shallow compartment molded from a drug - impermeable limited to , Carbopol, cellulose derivatives, bentonite , alg - laminate , such as a metallic plastic laminate, and a rate inates, gelatin and polyvinylpyrrolidone . 25 controlling polymeric membrane such as a microporous or a In addition to the active ingredient( s ), the ointments , nonporous polymeric membrane , e . g . , ethylene- vinyl acetate pastes , creams, and gels of the present invention can contain copolymer . The active ingredient is released through the excipients , such as animal and vegetable fats , oils , waxes , ratecontrolling polymeric membrane . In the drug reservoir, paraffins , starch , tragacanth , cellulose derivatives , polyeth - the active ingredient can either be dispersed in a solid ylene glycols , silicones , bentonites , silicic acid , talc and zinc 30 polymer matrix or suspended in an unleachable , viscous oxide, or mixtures thereof. liquid medium such as silicone fluid . On the external surface Powders and sprays can contain excipients such as lac of the polymeric membrane, a thin layer of an adhesive tose , talc , silicic acid , aluminum hydroxide , calcium silicates polymer is applied to achieve an intimate contact of the and polyamide powder, or mixtures of these substances. transdermal system with the skin surface . The adhesive Sprays can additionally contain customary propellants , such 35 polymer is preferably a polymer which is hypoallergenic and as chlorofluorohydrocarbons, and volatile unsubstituted compatible with the active drug substance . hydrocarbons , such as butane and propane . In an adhesive diffusion -controlled system , a reservoir of Injectable depot forms are made by forming microencap - the active ingredient is formed by directly dispersing the sule matrices of compound ( s ) of the invention in biodegrad active ingredient in an adhesive polymer and then by , e. g ., able polymers such as polylactide -polyglycolide . Depending 40 solvent casting , spreading the adhesive containing the active on the ratio of compound to polymer, and the nature of the ingredient ance onto a flat sheet of substantially drug particular polymer employed , the rate of compound release impermeable metallic plastic backing to form a thin drug can be controlled . Examples of other biodegradable poly - reservoir layer. mers include poly (orthoesters ) and poly (anhydrides ) . Depot A matrix dispersion - type system is characterized in that a injectable formulations are also prepared by entrapping the 45 reservoir of the active ingredient is formed by substantially drug in liposomes or microemulsions which are compatible homogeneously dispersing the active ingredient in a hydro with body tissue. philic or lipophilic polymer matrix . The drug -containing Subcutaneous implants are well - known in the art and are polymer is then molded into disc with a substantially well suitable for use in the present invention . Subcutaneous defined surface area and controlled thickness . The adhesive implantation methods are preferably non - irritating and 50 polymer is spread along the circumference to form a strip of mechanically resilient . The implants can be of matrix type , adhesive around the disc . of reservoir type , or hybrids thereof. In matrix type devices, A microreservoir system can be considered as a combi the carrier material can be porous or non - porous , solid or nation of the reservoir and matrix dispersion type systems. semi- solid , and permeable or impermeable to the active In this case, the reservoir of the active substance is formed compound or compounds . The carrier material can be bio - 55 by first suspending the drug solids in an aqueous solution of degradable or may slowly erode after administration . In water- soluble polymer and then dispersing the drug suspen some instances, the matrix is non -degradable but instead sion in a lipophilic polymer to form a multiplicity of relies on the diffusion of the active compound through the unleachable , microscopic spheres of drug reservoirs . matrix for the carrier material to degrade . Alternative sub - Any of the above - described controlled release , extended cutaneous implant methods utilize reservoir devices where 60 release , and sustained release compositions can be formu the active compound or compounds are surrounded by a rate lated to release the active ingredient in about 30 minutes to controlling membrane, e . g . , a membrane independent of about 1 week , in about 30 minutes to about 72 hours , in component concentration (possessing zero - order kinetics ). about 30 minutes to 24 hours , in about 30 minutes to 12 Devices consisting of a matrix surrounded by a rate con hours, in about 30 minutes to 6 hours, in about 30 minutes trolling membrane also suitable for use . 65 to 4 hours , and in about 3 hours to 10 hours . In embodi Both reservoir and matrix type devices can contain mate - ments , an effective concentration of the active ingredient (s ) rials such as polydimethylsiloxane, such as SilasticTM , or is sustained in a subject for 4 hours, 6 hours , 8 hours, 10 US 9 ,789 , 120 B2 113 114 hours , 12 hours , 16 hours , 24 hours , 48 hours , 72 hours , or is determined by first administering a low dose of the more after administration of the pharmaceutical composi - compound ( s ) and then incrementally increasing the admin tions to the subject. istered dose or dosages until a desired effect ( e . g . , decreased Methods of Delivery spermatozoa levels in seminal fluid ) is observed in the When the compound ( s ) of the invention are administered 5 treated subject, with minimal or acceptable toxic side as pharmaceuticals to humans and animals , they can be effects. Applicable methods for determining an appropriate given per se or as a pharmaceutical composition containing dose and dosing schedule for administration of a pharma active ingredient in combination with a pharmaceutically c eutical composition of the present invention are described , acceptable carrier, excipient, or diluent. for example , in Goodman and Gilman ' s The Pharmacologi Actual dosage levels and time course of administration of 10 cal Basis of Therapeutics, Goodman et al. , eds. , 11th Edi the active ingredients in the pharmaceutical compositions of tion , McGraw -Hill 2005 , and Remington : The Science and the invention can be varied so as to obtain an amount of the Practice of Pharmacy , 20th and 21st Editions, Gennaro and active ingredient which is effective to achieve the desired University of the Sciences in Philadelphia , Eds. , Lippencott therapeutic response for a particular patient, composition , & Wilkins ( 2003 and 2005 ) , which are hereby incorporated and mode of administration , without being toxic to the 15 by reference . patient. Generally , compounds or pharmaceutical composi Kits tions of the invention are administered in an effective The invention provides for a kit for effecting male con amount or quantity sufficient to reduce or inhibit spermato - traception . In embodiments , the kit contains one or more of zoa emission in a male subject. In embodiments , adminis - the compounds or pharmaceutical compositions described tration of the compound or pharmaceutical composition 20 herein . In embodiments , the kit provides instructions for use . suppresses spermatogenesis , induces azoospermia , or The instructions for use can pertain to any of the methods induces oligozoospermia . described herein . In related embodiments, the instructions Exemplary dose ranges include 0 .01 mg to 250 mg per pertain to using the compound ( s ) or pharmaceutical com day, 0 .01 mg to 100 mg per day, 1 mg to 100 mg per day, position (s ) for reducing or inhibiting spermatozoa emission . 10 mg to 100 mg per day , 1 mg to 10 mg per day , and 0 . 01 25 In embodiments , the kit provides a notice in the form mg to 10 mg per day . A preferred dose of the compound of prescribed by a governmental agency regulating the manu the invention is the maximum that a patient can tolerate and facture , use , or sale of pharmaceuticals or biological prod not develop serious or unacceptable side effects . In embodi - ucts, which notice reflects approval by the agency of manu ments, the compound ( s ) of the present invention is admin - facture , use or sale of the kit and the components therein for istered at a concentration of about 10 micrograms to about 30 human administration . 100 mg per kilogram of body weight per day, about 0 . 1 to The invention also provides for compound ( s ) or pharma about 10 mg/ kg per day, or about 1. 0 mg to about 10 mg/ kg ceutical composition (s ) packaged in a hermetically sealed of body weight per day. container ( e .g . , ampoule or sachette ) indicating the quantity In embodiments , the pharmaceutical composition com of compound . In embodiments , a compound or pharmaceu prises a compound ( s ) of the invention in an amount ranging 35 tical composition is supplied as a liquid . In other embodi between 1 and 10 mg, such as 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , or 10 ments , a compound or pharmaceutical composition is sup mg. plied as a dry sterilized lyophilized powder or water free In embodiments , the therapeutically effective dosage pro concentrate in a hermetically sealed container and can be duces a serum concentration of compound of from about 0 . 1 reconstituted , e . g . , with water or saline , to the appropriate ng /ml to about 50 - 100 ug /ml . The pharmaceutical compo - 40 concentration for administration to a subject. sitions typically should provide a dosage of from about The invention also provides for transdermal patches con 0 .001 mg to about 2000 mg of compound per kilogram oftaining the compound ( s ) or pharmaceutical composition ( s ) . body weight per day . For example , dosages for systemic In embodiments , the kit provides compound ( s ) or phar administration to a human patient can range from 1 - 10 maceutical composition ( s ) in more than one dosage unit . ug/ kg , 20 - 80 ug /kg , 5 - 50 ug/ kg , 75 - 150 ug /kg , 100 -500 45 The kit can contain from 1 to about 120 or more , from 1 to ug /kg , 250 - 750 ug /kg , 500 - 1000 ug /kg , 1 - 10 mg/ kg , 5 - 50 about 60 , from 1 to about 30 , from 1 to about 10 , or from 1 mg/ kg , 25 - 75 mg/ kg , 50 - 100 mg/ kg , 100 -250 mg/ kg , 50 - 100 to about 7 dosage units . In cases where the compound (s ) or mg/ kg , 250 -500 mg/ kg , 500 -750 mg/ kg , 750 - 1000 mg/ kg , pharmaceutical composition (s ) is adapted to release a thera 1000 - 1500 mg/ kg , 1500 - 2000 mg/ kg , 5 mg/ kg , 20 mg/ kg , 50 peutically effective amount of the active ingredient over a 24 mg/ kg , 100 mg/ kg , 500 mg/ kg , 1000 mg/ kg , 1500 mg/ kg , or 50 hour period , the kit conveniently comprises 1 , about 5 , about 2000 mg/ kg . Pharmaceutical dosage unit forms are prepared 7 , about 10 , about 14 , or about 30 dosage units . In cases to provide from about 1 mg to about 5000 mg, for example where the compound ( s ) or pharmaceutical composition ( s ) is from about 100 to about 2500 mg of the compound or a adapted to provide a therapeutically effective amount of the combination of essential ingredients per dosage unit form . active ingredient over a 12 hour period , the kit conveniently In embodiments , the pharmaceutical composition com - 55 comprises 1 , 2 , about 10 , about 14 , about 30 or about 60 prises a compound ( s ) of the invention in an amount suffi - dosage units . In cases where the compound ( s ) or pharma cient to lower spermatozoa concentration to not more than 3 ceutical composition ( s ) is adapted to provide a therapeuti million /mL of semen , such as not more than 2 million /mL , cally effective amount of the active ingredient over an about 1 million /mL , 0 . 5 million /mL , 0 .25 million /mL , or 0 . 1 3 to about 10 hour ( e . g ., about a 6 or 8 hour ) period , the kit million /mL . In related embodiments , the pharmaceutical 60 comprises about 1 , about 4 , about 40 , about 60 or about 120 composition comprises a compound (s ) of the invention in an dosage units . One skilled in the art will recognize that other amount sufficient to lower spermatozoa concentration to not numbers of dosage units can be included in the kit without more than 0 . 1 million /mL . departing materially from the present invention . Determination of an effective amount is well within the Screening Methods capability of those skilled in the art , especially in light of the 65 As described herein , the invention provides specific detailed disclosure provided herein . Generally , an effica - examples of chemical compounds , including JQ1, as well as cious or effective amount of a compound ( s ) of the invention other substituted compounds that bind a bromodomain bind US 9 , 789 ,120 B2 115 116 ing pocket and are useful as a male contraceptive . However, 1994 ; Cho et al . , Science 261: 1303 , 1993 ; Carrell et al . , the invention is not so limited . The invention further pro Angew . Chem . Int. Ed . Engl. 33 :2059 , 1994 ; Carell et al. , vides a simple means for identifying agents ( including Angew . Chem . Int. Ed . Engl. 33 : 2061, 1994 ; and Gallop et nucleic acids, peptides, small molecule inhibitors , and al. , J. Med . Chem . 37 : 1233 , 1994 . Furthermore , if desired , mimetics ) that are capable of inhibiting spermatogenesis . 5 any library or compound is readily modified using standard Such compounds are also expected to be useful as male chemical , physical , or biochemical methods. contraceptives . Numerous methods are also available for generating ran In particular embodiments , the effect of a compound or dom or directed synthesis ( e . g ., semi- synthesis or total other agent of the invention is analyzed by assaying sper matogenesis . Agents and compounds of the invention that 10 synthesis ) of any number of polypeptides, chemical com reduce spermatogenesis are identified as useful as male pounds , including , but not limited to , saccharide -, lipid -, contraceptives. peptide -, and nucleic acid -based compounds. Synthetic Virtually any agent that specifically binds to a BET family compound libraries are commercially available from Bran member or that reduces the biological activity of a BET don Associates (Merrimack , N . H . ) and Aldrich Chemical family member may be emploved in the methods of the 15 (Milwaukee , Wis . ). Alternatively , chemical compounds to invention . Methods of the invention are useful for the be used as candidate compounds can be synthesized from high -throughput low -cost screening of candidate agents that readily available starting materials using standard synthetic reduce or otherwise inhibit spermatogenesis . A candidate techniques and methodologies known to those of ordinary agent that specifically binds to a bromodomain of a BET skill in the art . Synthetic chemistry transformations and family member is then isolated and tested for activity in an 20 protecting group methodologies (protection and deprotec in vitro assay or in vivo assay for its ability to inhibit tion ) useful in synthesizing the compounds identified by the spermatogenesis . One skilled in the art appreciates that the methods described herein are known in the art and include , effects of a candidate agent on a cell is typically compared for example , those such as described in R . Larock , Com to a corresponding control cell not contacted with the prehensive Organic Transformations , VCH Publishers candidate agent. Thus, the screening methods include com - 25 ( 1989 ) ; T . W . Greene and P . G . M . Wuts, Protective Groups paring spermatogenesis in a testes contacted by a candidate in Organic Synthesis , 2nd ed ., John Wiley and Sons ( 1991 ) ; agent to the spermatogenesis present in an untreated control L . Fieser and M . Fieser. Fieser and Fieser ' s Reagents for testes . Organic Synthesis, John Wiley and Sons ( 1994 ) ; and L . Once identified , agents of the invention ( e . g . , agents that Paquette , ed ., Encyclopedia of Reagents for Organic Syn specifically bind to and /or antagonize a bromodomain ) may 30 the be used as male contraceptives . Potential bromodomainy 30 thesis, John Wiley and Sons ( 1995 ) , and subsequent editions antagonists include organic molecules, peptides, peptide thereof. mimetics, polypeptides , nucleic acid ligands, aptamers, and Libraries of compounds may be presented in solution antibodies that bind to a BET family member bromodomain ( e. g ., Houghten , Biotechniques 13: 412 -421 , 1992) , or on and reduce its activity . Candidate agents may be tested for 35 beads (Lam , Nature 354 : 82 - 84 , 1991) , chips (Fodor , Nature their ability to reduce spermatogenesis . 364 : 555 -556 , 1993 ) , bacteria (Ladner , U . S . Pat. No. 5, 223 , Test Compounds and Extracts 409 ) , spores (Ladner U . S . Pat. No. 5 ,223 ,409 ) , plasmids In certain embodiments , BET family member antagonists (Cull et al. , Proc Natl Acad Sci USA 89 : 1865 - 1869, 1992 ) or ( e .g . , agents that specifically bind and reduce the activity of on phage (Scott and Smith , Science 249 :386 - 390 , 1990 ; a bromodomain ) are identified from large libraries of natural 40 Devlin , Science 249 :404 -406 , 1990 ; Cwirla et al. Proc . Natl . product or synthetic ( or semi- synthetic ) extracts or chemical Acad . Sci . 87 :6378 -6382 , 1990 ; Felici, J . Mol. Biol. 222 : libraries or from polypeptide or nucleic acid libraries , 301 -310 , 1991; Ladner supra ) . according to methods known in the art . Those skilled in the In addition , those skilled in the art of drug discovery and field ofdrug discovery and development will understand that development readily understand that methods for dereplica the precise source of test extracts or compounds is not 45 tion ( e . g ., taxonomic dereplication , biological dereplication , critical to the screening procedure( s ) of the invention . Vir - and chemical dereplication , or any combination thereof ) or tually any number of unknown chemical extracts or comom the elimination of replicates or repeats of materials already pounds can be screened using the methods described herein . known for their activity should be employed whenever Examples of such extracts or compounds include, but are not possible . limited to , plant- , fungal -, prokaryotic - or animal -based 50 W extracts , fermentation broths, and synthetic compounds, as When a crude extract is found to have BET family well as the modification of existing polypeptides. member bromodomain binding activity further fractionation Libraries of natural polypeptides in the form of bacterial, of the positive lead extract is necessary to isolate molecular fungal , plant, and animal extracts are commercially avail constituents responsible for the observed effect. Thus , the able from a number of sources, including Biotics (Sussex , 55 goal of the extraction , fractionation , and purification process UK ) , Xenova ( Slough , UK ) , Harbor Branch Oceangraphics is the careful characterization and identification of a chemi Institute ( Ft. Pierce , Fla .) , and PharmaMar, U .S . A . (Cam cal entity within the crude extract that reduces spermato bridge , Mass . ) . Such polypeptides can be modified to genesisgenesis . MethodsMet of fractionation and purification of such include a protein transduction domain using methods known heterogenous extracts are known in the art. If desired , in the art and described herein . In addition , natural and 60 compounds shown to be useful as therapeutics are chemi synthetically produced libraries are produced , if desired , cally modified according to methods known in the art . according to methods known in the art, e . g . , by standard extraction and fractionation methods. Examples ofmethods EXAMPLES for the synthesis of molecular libraries can be found in the art, for example in : DeWitt et al. , Proc. Natl. Acad . Sci. 65 It should be appreciated that the invention should not be U . S . A . 90 :6909 , 1993 ; Erb et al. , Proc. Natl. Acad . Sci. USA construed to be limited to the examples that are now 91: 11422, 1994 ; Zuckermann et al. , J . Med . Chem . 37 : 2678 , described ; rather , the invention should be construed to US 9 , 789, 120 B2 117 118 include any and all applications provided herein and all -continued equivalent variations within the skill of the ordinary artisan . Me 1 ) NH2NH2, THF I. Chemical Examples HN f Me 0 ? 23° C . 2 ) CH3C ( OCH3) 3 , Me Toluene, 120° C . Synthesis and Methods of Preparation 85 % (2 -steps ) Compounds of the invention can be synthesized by meth - 10 ods described herein , and / or according to methods known to MeMé Me one of ordinary skill in the art in view of the description herein . S6

15 Me Scheme S1. Synthesis of the racemic bromodomain inhibitor ( + ) - JQ1. **111111 Me Me Me NC S , morpholine EtOH , 70°C. 20 70 % Me Me S1 NH2 Fmoc- Asp (Ot - Bu ) — OH 25 ( + )- JQ1 HCTU , i- Pr2NET DMF , 23° C . 90 % ( 2 - amino - 4 , 5 - dimethylthiophen - 3 - yl) ( 4 - chlorophe nyl) methanone ( S2 ) 30 The compound JQ1 was prepared according to the scheme ao shown above. COOt- Bu Sulfur ( 220 mg, 6 . 9 mmol, 1 .00 equiv ) was added as a Piperidine solid to a solution of 4 - chlorobenzoyl acetonitrile S1 ( 1 . 24 HN NHFmoc DMF, 23° C . 35 g , 6 . 9 mmol, 1 equiv ) , 2 -butanone (0 .62 ml, 6 . 9 mmol, 1 . 00 equiv ) , and morpholine (0 .60 ml, 6 . 9 mmol, 1 .00 equiv ) in 90 % ethanol (20 ml, 0 .35 M ) at 23° C .21 . The mixture was then heated to 70° C . After 12 hours, the reaction mixture was cooled to 23° C . and poured into brine ( 100 ml) . The 40 aqueous layer was extracted with ethyl acetate (3x50 ml) . Me Me The combined organic layers were washed with brine (50 ml) , were dried over anhydrous sodium sulphate , were filtered , and were concentrated under reduced pressure . The O residue was purified by flash column chromatography ( Com COOt- Bu 45 biflash RF system , 40 gram silica gel , gradient 0 to 100 % ethyl acetate - hexanes ) to afford S2 ( 1 . 28 g , 70 % ) as a yellow HN NH2 AcOH , ETOH 80° C . solid . 9595 % ( S ) - tert -Butyl - 3 - ( { [ (9H - fluoren - 9 - yl )methoxy ] 50 carbonyl} amino ) -4 - { [ 3 - ( 4 -chorobenzoyl ) - 4 , 5 - dim ethylthiophen - 2 - yl ] amino } - 4 -oxobutanoate (S3 ) Me Me ( 2 - (6 - Chloro - 1H -benzotriazole - 1 - yl ) - 1 , 1 , 3 , 3 - tetramethyl S4 aminium hexafluorophosphate (HCTU ) (827 mg, 2 . 0 mmol, SA 55 2 .00 equiv ), and N ,N -diisopropylethylamine (0 .72 ml, 4 .0 i mmol, 4 .00 equiv ) were added sequentially to a solution of Me P2S5, NaHCO3 9 - fluorenylmethoxycarbonyl- aspartic acid B - tert- butyl ester HN Me diglyme [Fmoc - Asp ( Ot- Bu ) -OH ] ( 864 mg, 2 . 1 mmol, 2 . 10 equiv ) in Me 85° C . N , N - dimethylformamide ( 1 . 5 ml, 1 . 0 M ) . The mixture was 65 % 60 then stirred at 23° C . for 5 min . S2 ( 266 mg, 1 . 0 mmol, 1 equiv ) was then added as a solid . The reaction mixture was stirred at 23° C . After 16 hours, ethyl acetate ( 20 ml) and brine ( 20 ml) were added . The two layers were separated , Me Me and the aqueous layer was extracted with ethyl acetate (2x20 65 ml) . The combined organic layers were washed with brine ( 30 ml) , were dried over with anhydrous sodium sulphate , $ were filtered , and were concentrated under reduced pressure . US 9 ,789 , 120 B2 119 120 The residue was purified by flash column chromatography toluene (6 ml, 0 . 06 M ) . The reaction mixture was heated to (Combiflash RF , 40 gram silica gel, gradient 0 to 100 % ethyl 120° C . After 2 h , all the solvents were removed under acetate -hexanes ) to afford S3 (625 mg, 90 % ) as brown oil. reduced pressure . The residue was purified by flash column chromatography (Combiflash system , 4 g silica gel, gradient (S ) - tert -butyl 3 -amino -4 -( ( 3 -( 4 -chlorobenzoyl ) -4 , 5 - 5 0 to 100 % ethyl acetate -hexanes ) to afford JQ1 ( 140 mg, dimethylthiophen - 2 - yl) amino ) - 4 -oxobutanoate (S4 ) 85 % in 2 steps ) as white solid . The reaction conditions further epimerized the stereogenic center , resulting in the Compound S3 (560 mg, 0 .85 mmol , 1 equiv ) was dis - racemate , JQ1 (determined with Berger Supercritical Fluid solved into 20 % piperidine in DMF solution ( 4 .0 ml, 0 .22 Chromatography (SFC ) with an AS - H column ). M ) at 23° C . After 30 min , ethyl acetate ( 20 ml) and brine 10 ( 20 ml) were added to the reaction mixture . The two layers were separated , and the aqueous layer was extracted with ethyl acetate ( 2x20 ml) . The combined organic layers were Scheme S2 . Synthesis of enantiomerically enriched ( + ) - JQ1. washed with brine ( 3x25 ml) , were dried over anhydrous NH2 sodium sulphate , were filtered , and were concentrated under 15 Fmoc - Asp ( Ot- Bu - OH reduced pressure . The residue was purified by flash column PyBOP, i - Pr2NE chromatography (Combiflash RF system , 24 gram silica gel, DMF, 23° C . gradient 0 to 100 % ethyl acetate -hexanes ) to afford free 72 % amine S4 ( 370 mg, 90 % ) as yellow solid . The enantiomeric purity dropped to 75 % ( determined with Berger Supercriti - 20 cal Fluid Chromatography (SFC ) using AS - H column ) . S2 ( S ) - tert- Butyl 2 - ( 5 - ( 4 - chlorophenyl) - 6 , 7 -dimethyl - 2 oxo -2 ,3 -dihydro -1H - thieno [ 2 ,3 -e ][ 1 ,4 ]diazepin - 3 -yl ) acetate (S5 ) 25 Amino ketone (S4 ) (280 mg, 0 .63 mmol) was dissolved in COOt- Bu 10 % acetic acid ethanol solution (21 ml, 0 .03 M ) . The 1111111 COOH Piperidine reaction mixture was heated to 85° C . After 30 minutes , all HN NHFmoc DMF , 23° C . solvents were removed under reduced pressure . The residue 30 90 % was purified by flash column chromatography ( Combiflash RF system , 12 gram silica gel, gradient 0 to 100 % ethyl acetate -hexanes ) to afford compound S5 (241 mg, 95 % ) as white solid . Enantiomeric purity of S5 was 67 % ( determined Me Me with Berger Supercritical Fluid Chromatography (SFC ) 35 using an AS - H column ) . S3 tert - Butyl 2 -( 5 -( 4 -chlorophenyl ) - 6 ,7 -dimethyl - 2 thioxo -2 ,3 -dihydro - 1H - thieno [ 2 ,3 - e ][ 1, 4 ]diazepin -3 yl) acetate ( S6 ) 40 Phosphorus pentasulfide (222 mg, 1 . 0 mmol, 2 . 00 equiv ) , COOt-Bu sodium bicarbonate (168 mg, 2 . 0 mmol, 4 .00 equiv ) were added sequentially to a solution of S5 (210 mg, 0 . 5 mmol, HN NH2 SiO2, Toluene 1 equiv ) diglyme ( 1 . 25 ml, 0 . 4M ) . The reaction mixture was 45 90° C . heated to 90° C . After 16 h , brine (20 ml) and ethyl acetate 95 % ( 35 ml) were added . The two layers were separated , and the aqueous layer was extracted with ethyl acetate ( 3x30 ml) . The combined organic layers were washed with brine ( 2x15 Me Me ml) , were dried over anhydrous sodium sulphate , were 50 filtered , and were concentrated under reduced pressure . The residue was purified by flash column chromatography (Com S4 biflash RF system , 24 gram silica gel , gradient 0 to 100 % ethyl acetate -hexanes ) to afford S6 (141 mg , 65 % ) as brown solid with recovered S5 ( 73 mg, 34 % ) . 55 tert- Butyl 2 - ( 4 - ( 4 - chlorophenyl) - 2 , 3 , 9 - trimethyl- 6H Bio Me KOt- Bu, THF , - 78 > - 10° C ., thieno [3 , 2 -f ][ 1 , 2, 4 ]triazolo [ 4, 3 -a ] [ 1, 4 ]diazepin - 6 -yl ) i - Me PO (OET ) 2C1, - 78 ? - 10° C . ; acetate [ ( + )JQ1 ] 60 Me CH3CONHNH2, n -BuOH , (0 .015 ml, 0 .45 mmol, 1 .25 equiv ) was added 90° C . to a solution of S6 ( 158 mg , 0 .36 mmol, 1 equiv ) in THF ( 2 .6 92 % ml, 0 . 14 M ) at 0° C . The reaction mixture was warmed to 23° C . , and stirred at 23° C . for 1 h . All solvents were MéMe Me removed under reduced pressure. The resulting hydrazine 65 was used directly without purification . The hydrazine was S5 then dissolved in a 2 : 3 mixture of trimethyl orthoacetate and US 9 ,789 , 120 B2 121 122 - continued afford compound S5 (168 mg, 95 % ) as white solid . Enantio meric purity of S5 was 90 % (determined with Berger N - N Supercritical Fluid Chromatography (SFC ) using an AS - H 111111 Me column ) . Me Me 5 ( S ) -tert - Butyl 2 - ( 4 - ( 4 - chlorophenyl) - 2 ,3 , 9 - trimethyl Me 6H -thieno [3 ,2 -f ] [1 , 2 ,4 ]triazolo [4 ,3 - a ][ 1, 4 ]diazepin 6 -yl ) acetate [ ( + ) JQ1 ] Potassium tert - butoxide ( 1 . 0 M solution in THF, 0 . 3 ml, 10 Me Me 10 0 . 30 mmol, 1 . 10 equiv ) was added to a solution of S5 ( 114 mg, 0 .27 mmol, 1 equiv ) in THF ( 1. 8 ml, 0 . 15 M ) at - 78° C . The reaction mixture was warmed to - 10° C ., and stirred ( + ) -JQ1 at 23° C . for 30 min . The reaction mixture was cooled to - 78° C . Diethyl chlorophosphate ( 0 .047 ml, 0 . 32 mmol, 15 1 . 20 equiv ) was added to reaction mixture 2 . The resulting mixture was warmed to - 10° C . over 45 min . Acetic ( S ) -tert - Butyl- 3 - { { [ ( 9H - fluoren - 9 - yl) methoxy ] hydrazide ( 30 mg, 0 .40 mmol, 1 . 50 equiv ) was added to carbonyl } amino ) -4 - { [3 - ( 4 -chlorobenzoyl ) - 4, 5 -dim reaction mixture . The reaction mixture was stirred at 23° C . ethylthiophen -2 -yl ] amino } - 4 -oxobutanoate (S3 ) After 1 h , 1 -butanol ( 2 .25 ml) was added to reaction mixture , which was heated to 90° C . After 1 h , all solvents were Du 20 removed under reduce pressure . The residue was purified (Benzotriazol - 1 - yloxyl )tripyrrolidinophosphonium (Py with flash column chromatography ( Combiflash system , 4g BOP ) (494 mg, 0 . 95 mmol, 0. 95 equiv ), N ,N -diisopropyl silica gel, gradient 0 to 100 % ethyl acetate - hexanes) to ethylamine ( 0 . 50 ml, 2 . 8 mmol, 2 . 75 equiv ) were added afford ( + ) -JQ1 (114 mg, 92 % ) as white solid with 90 % sequentially to a solution of 9 - fluorenylmethoxycarbonyl enantiomeric purity ( determined with Berger Supercritical aspartic acid p - tert - butyl ester [ Fmoc - Asp (Ot - Bu ) - OH ] (411 Fluid Chromatography (SFC ) using AS - H column , 85 % mg, 1 .00 mmol , 1. 0 equiv ) in N , N - dimethylformamide ( 1 . 0 25 hexanes -methanol . 210 nm , t . (R - enantiomer ) = 1 . 59 min , te ml, 1 . 0 M ) . The mixture was then stirred at 23° C . for 5 min . ( S - enantiomer ) = 3 .67 min ) . The product was further purified S2 ( 266 mg, 1 . 0 mmol, 1 equiv ) was then added as solid . The by chiral preparative HPLC (Agilent High Pressure Liquid reaction mixture was stirred at 23° C . After 4 h , ethyl acetate Chromatography using an OD - H column ) to provide the ( 20 ml) and brine (20 ml) were added . The two layers were S -enantiomer in greater than 99 % ee . separated , and the aqueous layer was extracted with ethyl 30 H NMR (600 MHz, CDC13 , 25° C .) 8 7. 39 (d , J= 8 . 4 Hz, acetate ( 2x20 ml) . The combined organic layers were 2H ), 7 .31 ( d , J = 8 . 4 Hz, 2H ) , 4 .54 ( t, J = 6 . 6 MHz, 1H ) , washed with brine , were dried over with anhydrous sodium 3 . 54 - 3 .52 ( m , 2H ) , 2 .66 ( s , 3H ) , 2 .39 ( s , 3H ), 1 .67 ( s, 3H ) , sulphate , were filtered , and were concentrated under reduced 148 ( s. 9 ) . pressure . The residue was purified by flash column chroma 13C NMR ( 150 MHz , CDC1z , 25° C . ) 8 171 . 0 , 163 . 8 , tography ( Combiflash RF system , 40 gram silica gel , gra - 1557. 150 . 0 1369 131. 1 . 130 . 9 130 6 1303. 1289 812 dient 0 to 100 % ethyl acetate -hexanes ) to afford S3 ( 152mg , 35 54 . 1 , 38 . 1 , 28 . 4 , 14 .6 , 13 . 5 , 12 . 1 . 72 % ) as brown oil . HRMS( ESI) calc ' d for C2 H24C1N2025 [ M + H ] * : ( S ) - tert - butyl 3 -amino - 4 - ( 3 - ( 4 -chlorobenzoyl ) - 4 , 5 457 .1460 , found 457 .1451 m /z . dimethylthiophen - 2 -yl ) amino ) -4 -oxobutanoate (S4 ) TLC ( EtOAC ) , Rf : 0 . 32 (UV ) 40 [ a ]? ? o = + 75 ( c 0 . 5 , CHC13 ) Compound S3 (310 mg, 0 .47 mmol, 1 equiv ) was dis - ( - ) - JQ1 was synthesized in a similar manner, employing solved into 20 % piperidine in DMF solution ( 2 .2 ml. 0 .22 Fmoc - D -Asp (Ot - Bu ) -OH as a starting material, and was M ) at 23° C . After 30 min , ethyl acetate ( 20 ml) and brine further purified by chiral preparative HPLC (Agilent High (20 ml) were added to the reaction mixture . The two layers Pressure Liquid Chromatography using an OD - H column ) to were separated , and the aqueous layer was extracted with 45 afford the R -enantiomer in greater than 99 % ee . [ a ] = - 72 ethyl acetate ( 2x20 ml) . The combined organic layers were c 0 .5 , CHC13 ) washed with brine ( 3x25 ml) , were dried over anhydrous Synthesis of Additional Compounds sodium sulphate , were filtered , and were concentrated under Additional compounds of the invention were prepared as reduced pressure . The residue was purified by flash column uatrotod chromatography (Combiflash RF system , 24 gram silica gel, 50 gradient 0 to 100 % ethyl acetate -hexane ) to afford free amine S4 (184 mg, 90 % ) as yellow solid . The enantiomeric Scheme 3 . Synthesis of hydrazine derivatives . purity was 91 % (checked with Berger Supercritical Fluid C1 Chromatography (SFC ) using an AS -H column ). 55 ( S )- tert- Butyl 2 -( 5 -( 4 -chlorophenyl ) - 6 , 7 -dimethyl - 2 oxo - 2 , 3 - dihydro - 1H -thieno [ 2 , 3 - e ] [ 1 , 4 ] diazepin - 3 - yl) acetate (S5 ) Amino ketone ( S4 ) ( 184 mg, 0 . 42 mmol ) was dissolved in 60 toluene ( 10 ml, 0 . 04 M ) . Silica gel ( 300 mg) was added , and the reaction mixture was heated to 90° C . After 3 h , the N reaction mixture was cooled to 23° C . The silica gel was -Z filtered , and washed with ethyl acetate . The combined fil trates were concentrated . The residue was purified by flash 65 column chromatography (Combiflash RF system , 12 gram ( 1 ) , ( + ) - JQ1 silica gel, gradient 0 to 100 % ethyl acetate -hexanes ) to US 9 , 789 , 120 B2 123 124 -continued - continued

u

- N - OH - 10 O Bonning OH EN

15 E As shown in Scheme S3 , the t -butyl ester of ( + ) - JQ1 ( 1 ) was cleaved to yield the free acid ( 2 ) , which was coupled with hydrazine to yield the hydrazide ( 3 ) . Reaction with 4 - hydroxybenzaldehyde yielded the ( 4 ) . Both hydrazide (3 ) and hydrazone (4 ) showed activity in at least one biological assay. NH2 A library of compounds was prepared by reaction of the 25 hydrazide ( 3 ) with a variety of carbonyl- containing com pounds (see Table A , above ). Additional compounds were prepared for use , e. g. , as probes for assay development. An exemplary synthesis is shown in Scheme S4 , below .

Scheme S4 . Synthesis of derivatives useful as probes.

MeOCOCI; HCOOH , 23° C . NH2NH2 Harrys85 % BarsOH 85 %

FITC , EtOH, 23° C . suit N 85 % NHNH2, N 8

Z US 9 ,789 , 120 B2 125 126 125 - continued

N NH CO2H

For FITC assay

EDC , HOBt, 23° C . OH 85 % O

1 ) 5 % TFA , CH2Cl2 , 95 % NHTrt 2 ) Biotin , EDC , HOBt, 23° C . termen90 %

HN NH

For Alpha assay US 9 , 789 , 120 B2 127 128 Additional compounds were prepared as shown in the table below :

Compound MS [M + H ]+ Name Structure m /z ( Observed )

( S ) -JQ1 N 457 . 1

( R ) - JQ1 457. 1

JQ3 starosta 415 . 1

III - NH NH2

JQ4 519 . 1

– NH US 9 ,789 , 120 B2 129 130 -continued Compound MS [ M + H ] * Name Structure m /z (Observed )

JQ6 493 . 1

Illll NH Non NH

JQ7 579 . 0

. IIIIII -NH

- SO2Na

JOS 494 . 1

Bull

NH NH

JQ10 501 . 1 US 9 ,789 , 120 B2 131 132 -continued Compound MS [ M + H ] + Name Structure m / z (Observed )

JQ11 F3C N 511 . 1 II lok

JO1- FITC OH 804 . 1

NNHNH CO2H

JQ1- Biotin C1 829 . 3

NH 1H

NH (PEC )7

O

JQ13 526 . 2 US 9, 789 ,120 B2 133 134 -continued Compound MS [ M + H ] * Name Structure m / z { Observed)

KS1 429. 1

JQ18 487, 1

llllll

Chemical Formula : CAH : - CINu03S Exact Mass : 486. 14924 Molecular Weight : 48701418

J019 471 . 1

- lllllll :

Chemical Formula : C24H27CIN402S Exact MaSS: 470. 15432 Molecular Weights 471 . 01478 US 9, 789 ,120 B2 135 136 -continued Compound MS [ M + H ] * Name Structure m ' z { Observed )

J020 370. 1

Chemical formmlula: CioHigCINAS Exact Mass : 370. 10190 Molecular Weight: 370. 89896 JQI- II - 023

JQ21 443 , 1

JQI- II - 024 Chemical Formula : C ? H ? ? CIN02S Exact MaSS: 44212303 Molecular Weight : 44296162

J024A 456, 1

Chemical Formula : C24H26CIN302S Exact Mass : 455 .1434 Molecular Weight : 458. 001 US 9, 789 ,120 B2 137 138 -continued Compound MS [ M + H ] * Name Structure m ' z { Observed )

J0248 456 , 1 1 <

Chemical Formula : C24H26CIN302S _ Exact Mass : 455. 1434 Molecular Weight : 458. 001

JQ25 506 , 1

HN

Chemical Formula : Ca « H24CIN302S Exact MaSS: 505. 1339 Molecular Weight: 506 . 0191

JQB 389. 2

Chemical Formula : C23H24N402 Exact Mass : 388 .1899 Molecular Weight: 388. 4623 ?

US 9, 789 ,120 B2 139 140 -continued Compound MS [ M + H ] * Name Structure m ' z { Observed )

J030 456. 2

- ANH

Chemical Formmlula : C23H cCINgOS _ Exact Mass : 455. 1547 Molecular Weight : 456. 0034

JQ31 456. 2

Chemical Formula : C2, HosCINgOS Exact Mass : 455. 1547 Molecular Weight : 456. 0034

JQ32 468. 1

F

Chemical Formula : CaHi- CIF ; NGOS _ Exact Mass : 467. 0794 Molecular Weight : 467. 8951 US 9 ,789 , 120 B2 141 142 -continued Compound MS [ M + H ] + Name Structure m /z (Observed )

JQ33 512 . 2

- NH

Chemical Formula : C25H29C1N202S Exact Mass : 512 .1761 Molecular Weight: 513 .0548

JQ34 505 . 1

- NH

Chemical Formula : C26H25CINGOS Exact Mass : 504 . 1499 Molecular Weight : 505. 0343

JQ35 540 . 2 hraNH

Chemical Formula : C27H34CINGOS Exact Mass : 539 .2234 Molecular Weight : 540 . 1232 US 9 , 789 ,120 B2 143 144 -continued Compound MS [ M + 1 ] Name Structure mz ( Observed )

JQ36 540 . 2

NHN

Chemical Formula : CH34CIN7OS Exact Mass: 539. 2234 Molecular Weight: 540. 1232

JQ37 424 . 2

Chemical Formula : C22H26N5OS Exact Mass : 423. 1729 ??????11Molecular Weight: 423. 5312 JQ38 508 . 2

- NH

Chemical Formula : C25H5CIN7OS Exact Mass : 507 . 1608 Molecular Weight: 508. 0362 US 9, 789 ,120 B2 145 146 -continued Compound MS [ M + H ] * Name Structure m / z ( Observed )

J039 505 , 1

Chemical Formula : CicH , , CIN & OS Exact MaSS: 504 . 1499 Molecular Weight: 505 . 0343

JQ40 514 . 2

IN HNHN

Chemical Formula : CasHI30CIN , OS ? Exact Mass : 511 .1921 Molecular Weight: 512. 0700

J041 540. 2

HN

Chemical Formula : C2 , H34CIN, OS Exact MaSS: 539. 2234 Molecular Weighit : 540. 1232 US 9, 789 ,120 B2 147 148 -continued

Compound MS [ K + H ] * Name Structure m / z { Observed) JQ42 441 . 2

z

Chemical Formula : C2 , H , 5FN402S Exact MaSS : 440. 1682 Molecular Weight: 440. 5336

JQ43 494 . 1

NH

Chemical Formiula : C24H24CIN - OS Exact IaSS : 493. 1452 Molecular Weight: 494. 011 ?

J044 513 , 2

N HN

Chemical Formula : C ?5H , , CIN, 02S Exact IaSS : 512 .1761 Molecular Weight: 513 . 0548 US 9 ,789 , 120 B2 149 150 -continued Compound MS [ M + H ] * Name Structure m /z (Observed )

JQ45 494 . 1

HN

Chemical Formula : C24H24CINGOS Exact Mass: 493 .1452 Molecular Weight : 494 .0117

JQ46 499 . 2

EN HN

Chemical Formula : C25H31CINGOS Exact Mass: 498 . 1969 Molecular Weight : 499 .0712

JQ47 ???? 626 . 3

EN

Chemical Formula : C32H44CIN ,02S Exact Mass : 625 . 2966 Molecular Weight: 626 . 2555 US 9 ,789 , 120 B2 151 152 - continued Compound MS [ M + H ] " Name Structure m /z (Observed ) JQ48 N — N 471 . 2

Exact Mass : 470 . 1543 Molecular Weight: 471. 0146

JQ49 429 . 1

Exact Mass : 428 . 1074 Molecular Weight: 428. 9350 JQ50 Byrna 540 . 2 Exact Mass : 539 .2234 Molecular Weight: 540 . 1232

JQ51 N - N 667. 2

ci JQI- II- 114

Exact Mass : 666 . 1816 Molecular Weight : 666 . 1764