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(12) United States Patent (10) Patent No.: US 9,301.962 B2 Bradner et al. (45) Date of Patent: Apr. 5, 2016
(54) MALE CONTRACEPTIVE COMPOSITIONS 5,854.238 A 12/1998 Kempen AND METHODS OF USE 6,444,664 B1 9, 2002 Princen et al. 7,015,213 B1 3/2006 Bigg et al. 7,589,167 B2 9, 2009 Zhou et al. (75) Inventors: James Elliott Bradner, Weston, MA 8,044,042 B2 * 10/2011 Adachi et al...... 514,219 (US); Martin Matzuk, Houston, TX 8,476,260 B2 * 7/2013 Miyoshi et al...... 514, 220 (US); Jun Qi, Sharon, MA (US) 8,981,083 B2 3/2015 Bradner et al. 2002fO169158 A1 1 1/2002 Hunt, III et al. (73) Assignees: Baylor College of Medicine, Houston, 2003. O130268 A1 7/2003 Sagara et al. 2006, O142257 A1 6/2006 Nieschlag et al. TX (US); Dana Farber Cancer 2006/0223055 A1 10/2006 Howley et al. Institute, Inc., Boston, MA (US) 2007/0218135 A1 9/2007 Mukharya et al. 2008.0004308 A1 1/2008 Dhanak et al. (*) Notice: Subject to any disclaimer, the term of this 2008/0081781 A1 4/2008 Lippa et al. patent is extended or adjusted under 35 2009/0012064 A1 1/2009 Sagara et al. U.S.C. 154(b) by 0 days. 2010/0286127 A1 1 1/2010 Miyoshi et al. 2012/0202798 A1 8/2012 Sagara 2013, O184264 A1 7/2013 Bradner et al. (21) Appl. No.: 13/698,006 2013,0252331 A1 9/2013 Bradner et al. (22) PCT Filed: May 16, 2011 2014/0011862 A1 1/2014 Bradner et at. (86). PCT No.: PCT/US2O11AO366.67 FOREIGN PATENT DOCUMENTS CA 2020806 A1 1, 1991 S371 (c)(1), CA 271074.0 A1 T 2009 (2), (4) Date: Apr. 26, 2013 CH 622O19 A5 3, 1981 CN 1227.555 A 9, 1999 (87) PCT Pub. No.: WO2011/143657 DE 3724164 A1 2, 1988 EP O 087 850 A1 9, 1983 PCT Pub. Date: Nov. 17, 2011 EP O 368 175 A1 5, 1990 EP O 387 613 A1 9, 1990 (65) Prior Publication Data EP O 93494.0 A1 8, 1999 EP O 989 131 A1 3, 2000 US 2013/0210813 A1 Aug. 15, 2013 EP 1297 836 A1 4/2003 EP 2 239264 A1 10, 2010 Related U.S. Application Data FR T532815 A1 5, 1977 JP 1-299231 12/1989 (60) Provisional application No. 61/334,991, filed on May JP 6-157316 6, 1994 14, 2010, provisional application No. 61/370,745, JP 11-228576 8, 1999 JP 3OO1979 11, 1999 filed on Aug. 4, 2010, provisional application No. JP 3096,299 8, 2000 61/375,863, filed on Aug. 22, 2010, provisional WO WO 97/47622 A1 12/1997 application No. 61/467,376, filed on Mar. 24, 2011, WO WO 98.11111 A1 3, 1998 provisional application No. 61/467,299, filed on Mar. WO WOO1,959 12 A1 12/2001 24, 2011. WO WO 2008/083056 A2 T 2008 WO WO 2008/137081 A1 11, 2008 WO WO 2009/084693 A1 T 2009 (51) Int. Cl. WO WO 2010/O15387 A1 2, 2010 AOIN 43/00 (2006.01) WO WO 2010/04946.6 A1 5, 2010 A 6LX3/553 (2006.01) A6 IK3I/554 (2006.01) (Continued) A6 IK3I/55 (2006.01) CO7D 495/4 (2006.01) OTHER PUBLICATIONS A 6LX3/557 (2006.01) Patani et al. Bioisosterism: A Rational Approach to Drug Design, (52) U.S. Cl. Chemical Reviews, 1996, vol. 96, No. 8.* CPC ...... A61K3I/551 (2013.01); A61 K3I/5517 (2013.01); C07D 495/14 (2013.01) (Continued) (58) Field of Classification Search None See application file for complete search history. Primary Examiner — Craig Ricci Assistant Examiner — Jared D Barsky (56) References Cited (74) Attorney, Agent, or Firm — Foley Hoag LLP U.S. PATENT DOCUMENTS 3,681,343 A 8, 1972 Hester, Jr. (57) ABSTRACT 3,709,898 A 1/1973 Hester, Jr. 3,812,259 A 5, 1974 Collins The invention relates to compositions and methods for effect 4,621,083. A 11, 1986 Casals-Stenzel et al. 5,104.543 A 4, 1992 Brandt et al. ing male contraception. 5,593.988 A 1/1997 Tahara et al. 5,712,274 A * 1/1998 Sueoka et al...... 514,219 5,753,649 A 5, 1998 Tahara et al. 22 Claims, 19 Drawing Sheets US 9,301.962 B2 Page 2
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This application is the U.S. National Stage of International Application No. PCT/US2011/036667, filed May 16, 2011, SUMMARY OF THE INVENTION which designates the U.S., published in English, and claims the benefit of U.S. Provisional Application No. 61/334,991, 10 As described below, this invention provides novel com filed May 14, 2010, U.S. Provisional Application No. 61/370, pounds and compositions for effecting male contraception. 745, filed Aug. 4, 2010, U.S. Provisional Application No. The invention also provides methods for using such com 61/375,863, filed Aug. 22, 2010, U.S. Provisional Applica pounds and compositions in a male Subject. tion No. 61/467,376, filed Mar. 24, 2011, and U.S. Provi In one aspect, the invention provides methods for reducing sional Application No. 61/467,299, filed Mar. 24, 2011. The 15 or inhibiting spermatogenesis in a male Subject. In embodi enire teachings of the above applications are incorporated ments, the methods involve administering an effective herein by reference. amount of a compound or a salt thereofthat inhibits abromo domain testis-specific protein (BRDT) to the male subject. Incorporation. By Reference Of Material In AscII In one aspect, the invention provides methods for reducing Text File the rate of male fertility in a subject. In embodiments, the methods involve administering an effective amount of a com This application incorporates by reference the Sequence pound or a salt thereof that inhibits a BRDT to the male Listing contained in the following ASCII text file being sub Subject. mitted concurrently herewith: In the above aspects, the methods involve administering a) File name: 48051003002sequence.txt; created Apr. 24, 25 the compound or a salt thereof in an amount Sufficient to 2013, 30 KB in size. reduce sperm number and/or reduce sperm motility. In the above aspects, the methods involve administering STATEMENT OF RIGHTS TO INVENTIONS the compound or a salt thereof in an amount Sufficient to MADE UNDER FEDERALLY SPONSORED induce azoospermia, oligoZoospermia, and/or asthenoZo RESEARCH 30 ospermia. In embodiments, the methods induce a contracep tive effect in the subject. This work was supported by the following grant from the In aspect of the invention, the invention provides pharma National Institutes of Health, Grant No: K08CA128972 ceutical compositions having a compound that inhibits (Bradner). The government has certain rights in the invention. 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 reduce traceptive drugs for men have remained elusive. To date, the 40 sperm motility. only drugs in clinical trials are testosterone 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 of unplanned pregnancies, especially in teenagers, and the compound or a salt thereof is present in a amount effective to associated maternal mortality and ethical, Social, and finan 45 induce a contraceptive effect in the Subject. cial costs associated with abortions and deliveries to single In any of the above aspects, the compound or a salt thereof mothers. To approach this dearth of contraceptive alternatives is administered to the Subject using any dosage and/or route of for men, it is desirable to develop small molecules that could administration described herein. In embodiments, the com target spermatogenic-specific proteins that have been shown pound or a salt thereof is administered to the subject orally, to be essential for both spermatogenesis and fertility in mam 50 transdermally, or by injection. In related embodiments, the mals. One such contraceptive target is the bromodomain tes compound or a salt thereof is administered in the form of a tis-specific protein, BRDT. tablet or capsule. In related embodiments, the compound or a BRDT is a tissue-restricted, chromatin remodeling protein salt thereof is administered by parenteral injection, intramus expressed in pachytene spermatocytes, diplotene spermato cular injection, intravenous injection, Subcutaneous implan cytes, and round spermatids. During post-meiotic maturation, 55 tation, Subcutaneous injection, or transdermal preparation. BRDT localizes to the nucleus and reorganizes hyperacety In any of the above aspects, the compound or a salt thereof lated histones through twin acetyl-lysine recognition mod is administered in combination with a pharmaceutically ules, or bromodomains. The essential role of BRDT in sper acceptable carrier, excipient, or diluent. matogenesis is mediated by the first bromodomain (BRDT In any of the above aspects, administration of the com (1)), which binds the tetra-acetylated amino-terminal tail of 60 pound or a salt thereof reduces epididymal sperm number by histone 4 (H4Kac4) with moderate potency (20 uM). Struc at least about 25% of the sperm number present in a control. tural studies of murine BRDT have demonstrated that BRDT In embodiments, administration of the compound or a salt (1) binds a diacetylated histone 4 peptide (H4K5ac8ac) in thereof reduces epididymal sperm number by at least about part through a conserved asparagine, akin to seminal studies 10% of the sperm number present in a control. In related of other bromodomain co-activator proteins. Genetic studies 65 embodiments, only about 5% of the spermatozoa remaining of BRDT have demonstrated that selective deletion of the show progressive motility after administration of the com BRDT(1)-encoding region is sufficient to confer sterility in pound or a salt thereof. US 9,301.962 B2 3 4 In any of the above aspects, administration of the com nal fluid during discharge of the seminal fluid from a male pound or a salt thereoflowers the spermatozoa concentration subject. Reduction or inhibition of spermatozoa levels in to not more than 3 million/mL, 2 million/mL, 1 million/mL, seminal fluid can be effected by Suppressing spermatogen 0.5 million/mL, 0.25 million/mL, or 0.1 million/mL. In related embodiments, administration of the compound or a esis, inducing azoospermia, inducing oligoZoospermia, and salt thereoflowers the spermatozoa concentration to not more the like. Thus, in the context of the present invention, “reduc than 0.1 million/mL. ing or inhibiting spermatozoa emission' has the effect of In one aspect, the invention provides kits for reducing male inhibiting and/or reducing the rate of fertilization when the fertility. In embodiments, the kits contain an effective amount of a compound that inhibits BRDT or a pharmaceutically discharged seminal fluid contacts ova from a female Subject. acceptable salt or prodrug thereof. In embodiments, the kits 10 “Spermatogenesis' refers to the overall process of game contain instructions for using the inhibitor in any of the meth- togenesis in the male. Spermatogenesis takes place in the od deters h d is JO1 seminiferous tubule and is directly regulated by levels of in any of the above aspects, the compound is JO1 or a follicle stimulating hormone and androgen at the periphery of compound of any of Formulas I-XXII, or any compound th inif tubul rticularl the Sertoli cell disclosed herein, or a derivative or salt thereof. In embodi- e semin1Ierous tubule, part1cularly upon une Sertol1 cells. ments, the compoundis JQ1 or a pharmaceutically acceptable The term "azoospermia” refers to a spermatozoa content salt or prodrug thereof. below 1 million per mL seminal fluid, approaching levels of In any of the above aspects, the compound or a Salt thereof zerospermatozoa content, and are the result of suppression of is administered in combination with at least one additional Spermatogenesis. additionalmale contraceptive male contraceptive agent or isdevice. a condom. In embodiments, In embodiments, the 20. The term &Goligozoospermia 1. 99 refers to a spermatozoa con the additional male contraceptive is a modulator of testoster- tent between 20 and one million per mL (mill/mL) seminal one production, androgen receptor function or stability. fluid, and are the result of inhibited levels of spermatogenesis. setAdditional forth in part objects in the and description advantages which of the follows, invention and willin part be By & Gbromodomain 99 1S meantaportion ofa polypeptide that will be obvious from the description, or may be learned by is recognizes acetylated lysine residues. In one embodiment, a practice of the invention. The objects and advantages of the bromodomain of a BET family member polypeptide com invention will berealized and attained by means of the ele- prises approximately 110 amino acids and shares a conserved ments and combinations disclosed herein, including those fold left-handed bundle off lpha heli pointed out in the appended claims. It is to be understood that o comprising a T- an e unt O our alpha he ices both the foregoing general description and the following linked by diverse loop regions that interact with chromatin. detailed description are exemplary and explanatory only and 30 & G 99 are not restrictive of the invention as claimed. The accompa- By BET family polypeptide 1S meanta polypeptide com nying drawings, which are incorporated in and constitute a prising two bromodomains and an extraterminal (ET) domain part of this specification, illustrate several embodiments of or a fragment thereofhaving transcriptional regulatory activ the invention and, together with the description, serve to ity or acetylated lysine binding activity. Exemplary BET fam explain the principles of the invention. ily members include BRD2, BRD3, BRD4 and BRDT. Definitions By "BRD2 polypeptide' is meant a protein or fragment To facilitate- an understanding of the present invention, a thereof having- at least 85% identity to NP 005095 that is number of terms and phrases are defined below capable of binding chromatin or regulating transcription. The term “reducing or inhibiting spermatozoa emission' The sequence (SEQ ID NO: 1) of an exemplary BRD2 refers to lowering the amount of spermatozoa presentin semi- polypeptide follows:
MLONWTPHNKLPGEGNAGLLGLGPEAAAPGKRIRKPSLLYEGFESPTMASVPALOLTPA
NPPPPEVSNPKKPGRVTNOLOYLHKVWMKALWKHOFAWPFROPVDAVKLGLPDYHKIIKH
OPMDMGTIKRRLENNYYWAASECMODFNTMFTNCYIYNKPTDDIVLMAQTLEKIFLOKW
ASMPOEEOELVVTIPKNSH KKGAKLAALQGSVTSA HOV PAWSSWSHTALYTPPPEI pTT
WLNIPHPSWISSPLLKSLHSAGPPLLAVTAAPPAQ PLA KKKGWKRKADTTTPTPTAILA
PGSPASPPGSLEPKAA RLP PMRRESGRPIKPPRKD LPDSQQOHOSSKKGKLSEOLK HCN
GILKELLSKKHAAYAW KPWDASALGLHDYHDIIKH PMDLSTWKRKMENRDYRDAOE
FAADWRLMFSNCYKYN PPD HDVVAMARKLODWFEF RYA KMPDEPLEPGPLPWSTAM PPG
LAKSSSESSSEESSSESSSEEEEEEDEEDEEEEESESS DSEEERAHRLAELQEOLRAVH
EOLAALSQGPISKPKR KRE KKEKKKKRKAEKHRGRAGA DEDDKGPRAPRPPOPKKS KKA
SGSGGGSAALGPSGFG KATKTAPPALPTGYDSEEEEESRPMSYDE
LSLDINKLPGEKLGRVWHIIQAREPSL RDSNPEEIEID FETLKPSTLRELERYWLSCLR
KEELALEKKRE LNSTKKPPKKANEKTESSSAO
QWAVSRLSASSSSSDSSSSSSSSSSSDTSDSDSG
US 9,301.962 B2 7 8 By “Brd4 nucleic acid molecule' is meanta polynucleotide half-life, without altering, for example, ligand binding. An that encodes a BRD4 polypeptide. analog may include an unnatural amino acid. By "BRDT polypeptide is meant a protein or fragment As used herein, the term “alkyl means a saturated Straight thereofhaving at least 85% identity to NP 001717 (SEQ ID chain or branched non-cyclic hydrocarbon typically having NO: 4) that is capable of binding chromatin or regulating 5 from 1 to 10 carbon atoms. Representative saturated straight transcription. chain alkyls include methyl, ethyl, n-propyl. n-butyl, n-pen
mslps rotai iv.nppppeyi intkkingriltin gloylqkvvl kdlwkhsfsw pfgrpvdavk 61 lolpdyyti i knpmdlintik krilenkyyak a seciedfnt mifsncylynk, pgddivlmaq 121 aleklfmokil sqmpqeeqvv gvkerikkgt qqniayssak eks spsatek vifikodeips v 181 fpktsi splin vvogasvins's sqtaaqvtkg vkirkadtttp at savkasse fsptfteksv 241 alppilkenmp knvlpdisqqq yinvvktvkvt eqlrhc seil kemlakkhfs yawpfyinpvd 3 O1 vnalglhnyy divvknpmdlig tikekmdnqe ykdaykfaad virlmfmncyk ynppdhevvt 3 61 marmlqdvfe thfskipiep vesmplcylik toitettigre intineassegn ssdds ederv 421 kirlaklqeql kavhqqldvil sqvpfrklink kkeksikkekk kekvinnsnen prkmc eqmrl 481 kekskrincipk kirkcgfiglk sedednakpm nydekrols1 ninklpgdkl grvivhiiqsr 541 epsils.nsnpd eieidfetlik astlireleky visa clirkirpl kppakkimms keelhsqkko 601 elekrilldvin nglin Srikrot ksdktopska Venvisirlses sssssssses ess ssdlsss 661 dissose semif pkftevkpnd spskenvkkm knecilpeg|r tdvtdigy cv qdtts antitl 721 vhottpshvm ppnhhdlafin yoelehlqtv knisplgilp psgdseqlsn gitvmhpsgd 781 sottmlesec gapvckdiki knadswkslg kpvkpsgwmk ssdelfinq.fr kaaiekevka 841 rtgelirkhl eqntkelikas gencirdligng litvesfsniki gnkcs geegk ehdos Seaqd 901. kskilwllkdr dilarqkeder rrreamvgti dimtlqsdimt mifennfa
By “BRDT nucleic acid molecule' is meant a polynucle- 35 tyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl; while otide encoding a BRDT polypeptide. saturated branched alkyls include isopropyl. Sec-butyl, isobu Administering is defined herein as a means of providing tyl, tert-butyl, isopentyl, 2-methylbutyl, 3-methylbutyl, an agent to a Subject in a manner that results in the agent being 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methyl inside the subject’s body. Such an administration can be by hexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2.3- any route including, without limitation, oral, transdermal, 40 dimethylbutyl, 2,3-dimethylpenty1, 2,4-dimethylpenty1, 2,3- mucosal (e.g., vagina, rectum, oral, or nasal mucosa), by dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2.2- injection (e.g., Subcutaneous, intravenous, parenterally, intra dimethylpenty1, 2,2-dimethylhexyl, 3.3-dimethylpentyl, 3.3- peritoneally, intrathecal), or by inhalation (e.g., oral or nasal). dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl, Pharmaceutical preparations are given by forms suitable for 3-ethylpentyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 45 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, 2-methyl the desired route of administration. 4-ethylpentyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethyl By "agent” or “compound is meant any small molecule hexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpenty1,3,3-diethyl chemical compound, antibody, nucleic acid molecule, or hexyl, 2,2-diethylhexyl, 3.3-diethylhexyl and the like. Alkyl polypeptide, or fragments thereof. groups included in compounds of this invention may be By “ameliorate' is meant decrease, Suppress, attenuate, 50 unsubstituted, or optionally substituted with one or more diminish, arrest, or stabilize the development or progression Substituents. Such as amino, alkylamino, arylamino, het of a disease. eroarylamino, alkoxy, alkylthio, oxo, halo, acyl, nitro, By “alteration' is meant a change (increase or decrease) in hydroxyl, cyano, aryl, heteroaryl, alkylaryl, alkylheteroaryl, the expression levels or activity of a gene or polypeptide as aryloxy, heteroaryloxy, arylthio, heteroarylthio, arylamino, detected by standard art known methods such as those 55 heteroarylamino, carbocyclyl carbocyclyloxy, carbocy described herein. As used herein, an alteration includes a 10% clylthio, carbocyclylamino, heterocyclyl, heterocyclyloxy, change in expression levels, preferably a 25% change, more heterocyclylamino, heterocyclylthio, and the like. Lower preferably a 40% change, and most preferably a 50% or alkyls are typically preferred for the compounds of this inven greater change in expression levels. tion. By “analog is meant a molecule that is not identical, but 60 As used herein, the term an “aromatic ring or “aryl has analogous functional or structural features. For example, means a monocyclic or polycyclic-aromatic ring or ring radi a polypeptide analog retains at least some of the biological cal comprising carbon and hydrogen atoms. Examples of activity of a corresponding naturally-occurring polypeptide, Suitable aryl groups include, but are not limited to, phenyl, while having certain biochemical modifications that enhance tolyl, anthacenyl, fluorenyl, indenyl, aZulenyl, and naphthyl, the analog's function relative to a naturally occurring 65 as well as benzo-fused carbocyclic moieties such as 5,6,7,8- polypeptide. Such biochemical modifications could increase tetrahydronaphthyl. An aryl group can be unsubstituted or the analogs protease resistance, membrane permeability, or optionally is substituted with one or more Substituents, e.g., US 9,301.962 B2 10 Substituents as described herein for alkyl groups (including a candidate compound. For example, a computer modeling without limitation alkyl (preferably, lower alkyl or alkyl sub experiment can compare a pharmacophore model of the stituted with one or more halo), hydroxy, alkoxy (preferably, invention with a candidate compound to assess the fit of the lower alkoxy), alkylthio, cyano, halo, amino, boronic acid candidate compound with the model. (—B(OH), and nitro). In certain embodiments, the aryl By a "computer system’ is meant the hardware means, group is a monocyclic ring, wherein the ring comprises 6 Software means and data storage means used to analyse carbon atoms. atomic coordinate data. The minimum hardware means of the The term "diastereomers' refers to stereoisomers with two computer-based systems of the present invention comprises a or more centers of dissymmetry and whose molecules are not central processing unit (CPU), input means, output means minor images of one another. 10 The term “enantiomers' refers to two stereoisomers of a and data storage means. Desirably a monitor is provided to compound which are non-Superimposable minor images of visualise structure data. The data storage means may be RAM one another. An equimolar mixture of two enantiomers is or means for accessing computer readable media of the inven called a “racemic mixture' or a “racemate.” tion. Examples of such systems are microcomputer worksta The term “halogen designates —F. —Cl, Br or —I. 15 tions available from Silicon Graphics Incorporated and Sun The term “haloalkyl is intended to include alkyl groups as Microsystems running Unix based, Windows NT or IBM defined above that are mono-, di- or polysubstituted by halo OS/2 operating systems. gen, e.g., fluoromethyl and trifluoromethyl. By “computer readable media' is meant any media which The term “hydroxyl means —OH. can be read and accessed directly by a computer e.g. So that The term "heteroatom' as used herein means an atom of the media is suitable for use in the above-mentioned computer any element other than carbon or hydrogen. Preferred het system. The media include, but are not limited to: magnetic eroatoms are nitrogen, oxygen, Sulfur and phosphorus. storage media such as floppy discs, hard disc storage medium The term "heteroaryl refers to an aromatic 5-8 membered and magnetic tape; optical storage media such as optical discs monocyclic, 8-12 membered bicyclic, or 11-14 membered or CD-ROM; electrical storage media such as RAM and tricyclic ring system having 1-4 ring heteroatoms if monocy 25 ROM; and hybrids of these categories such as magnetic/ clic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricy optical storage media. clic, said heteroatoms selected from O, N, or S, and the In this disclosure, "comprises.” “comprising.” “contain remainder ring atoms being carbon. Heteroaryl groups may ing” and “having and the like can have the meaning ascribed be optionally Substituted with one or more Substituents, e.g., to them in U.S. patent law and can mean “includes.” “includ Substituents as described herein foraryl groups. Examples of 30 ing.” and the like: “consisting essentially of or “consists heteroaryl groups include, but are not limited to, pyridyl, essentially” likewise has the meaning ascribed in U.S. patent furanyl, benzodioxolyl, thienyl, pyrrolyl, oxazolyl, oxadiaz law and the term is open-ended, allowing for the presence of olyl, imidazolyl, thiazolyl, isoxazolyl, quinolinyl, pyrazolyl, more than that which is recited so long as basic or novel isothiazolyl pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, characteristics of that which is recited is not changed by the triazolyl, thiadiazolyl, isoquinolinyl, indazolyl, benzox 35 presence of more than that which is recited, but excludes prior azolyl, benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl, art embodiments. benzimidazolyl, benzothiazolyl, benzothiadiazolyl, ben “Detect” refers to identifying the presence, absence or Zoxadiazolyl, and indolyl. amount of the analyte to be detected. The term "heterocyclic” as used herein, refers to organic By “detectable label' is meant a composition that when compounds that contain at least at least one atom other than 40 linked to a molecule of interest renders the latter detectable, carbon (e.g., S.O., N) within a ring structure. The ring struc via spectroscopic, photochemical, biochemical, immu ture in these organic compounds can be either aromatic or, in nochemical, or chemical means. For example, useful labels certain embodiments, non-aromatic. Some examples of het include radioactive isotopes, magnetic beads, metallic beads, erocyclic moeities include, are not limited to, pyridine, pyri colloidal particles, fluorescent dyes, electron-dense reagents, midine, pyrrolidine, furan, tetrahydrofuran, tetrahy 45 enzymes (for example, as commonly used in an ELISA), drothiophene, and dioxane. biotin, digoxigenin, or haptens. The term "isomers' or “stereoisomers' refers to com By “effective amount' is meant the amount of an agent pounds which have identical chemical constitution, but differ required to provide contraception to anotherwise fertile male. with regard to the arrangement of the atoms or groups in The effective amount of active compound(s) used to practice Space. 50 the present invention for therapeutic treatment of a disease The term “isotopic derivatives” includes derivatives of varies depending upon the manner of administration, the age, compounds in which one or more atoms in the compounds are body weight, and general health of the subject. Ultimately, the replaced with corresponding isotopes of the atoms. For attending physician or veterinarian will decide the appropri example, an isotopic derivative of a compound containg a ate amount and dosage regimen. Such amount is referred to as carbon atom (C) would be one in which the carbonatom of 55 an “effective” amount. the compound is replaced with the C' isotope. The term “enantiomers' refers to two stereoisomers of a By "computer modeling' is meant the application of a compound which are non-Superimposable minor images of computational program to determine one or more of the fol one another. An equimolar mixture of two enantiomers is lowing: the location and binding proximity of a ligand to a called a “racemic mixture' or a “racemate.” binding moiety, the occupied space of a bound ligand, the 60 The term “halogen designates —F. —Cl, Br or —I. amount of complementary contact surface between a binding The term “haloalkyl is intended to includealkyl groups as moiety and a ligand, the deformation energy of binding of a defined above that are mono-, di- or polysubstituted by halo given ligand to a binding moiety, and some estimate of hydro gen, e.g., fluoromethyl and trifluoromethyl. genbonding strength, Vander Waals interaction, hydrophobic The term “hydroxyl means —OH. interaction, and/or electrostatic interaction energies between 65 The term "heteroatom' as used herein means an atom of ligand and binding moiety. Computer modeling can also pro any element other than carbon or hydrogen. Preferred het vide comparisons between the features of a model system and eroatoms are nitrogen, oxygen, Sulfur and phosphorus. US 9,301.962 B2 11 12 The term "heteroaryl refers to an aromatic 5-8 membered prokaryote or eukaryote; or that exists as a separate molecule monocyclic, 8-12 membered bicyclic, or 11-14 membered (for example, a cDNA or a genomic or cDNA fragment pro tricyclic ring system having 1-4 ring heteroatoms if monocy duced by PCR or restriction endonuclease digestion) inde clic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricy pendent of other sequences. In addition, the term includes an clic, said heteroatoms selected from O, N, or S, and the RNA molecule that is transcribed from a DNA molecule, as remainder ring atoms being carbon. Heteroaryl groups may well as a recombinant DNA that is part of a hybrid gene be optionally substituted with one or more substituents. encoding additional polypeptide sequence. Examples of heteroaryl groups include, but are not limited to, By an "isolated polypeptide' is meant a polypeptide of the pyridyl, furanyl, benzodioxolyl, thienyl, pyrrolyl, oxazolyl, invention that has been separated from components that natu oxadiazolyl, imidazolyl thiazolyl, isoxazolyl, quinolinyl, 10 rally accompany it. Typically, the polypeptide is isolated pyrazolyl, isothiazolyl pyridazinyl, pyrimidinyl, pyrazinyl, when it is at least 60%, by weight, free from the proteins and triazinyl, triazolyl, thiadiazolyl, isoquinolinyl, indazolyl, naturally-occurring organic molecules with which it is natu benzoxazolyl, benzofuryl, indolizinyl, imidazopyridyl, tetra rally associated. Preferably, the preparation is at least 75%, Zolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, more preferably at least 90%, and most preferably at least benzoxadiazolyl, and indolyl. 15 99%, by weight, a polypeptide of the invention. An isolated The term "heterocyclic” as used herein, refers to organic polypeptide of the invention may be obtained, for example, by compounds that contain at least at least one atom other than extraction from a natural Source, by expression of a recom carbon (e.g., S.O., N) within a ring structure. The ring struc binant nucleic acid encoding Such a polypeptide; or by chemi ture in these organic compounds can be either aromatic or cally synthesizing the protein. Purity can be measured by any non-aromatic. Some examples of heterocyclic moeities appropriate method, for example, column chromatography, include, are not limited to, pyridine, pyrimidine, pyrrolidine, polyacrylamide gel electrophoresis, or by HPLC analysis. furan, tetrahydrofuran, tetrahydrothiophene, and dioxane. By "marker is meant any protein or polynucleotide having The term "isomers' or “stereoisomers' refers to com an alteration in expression level or activity that is associated pounds which have identical chemical constitution, but differ with a disease or disorder. with regard to the arrangement of the atoms or groups in 25 As used herein, "obtaining as in "obtaining an agent' Space. includes synthesizing, purchasing, or otherwise acquiring the The term “isotopic derivatives” includes derivatives of agent. compounds in which one or more atoms in the compounds are The term “obtaining as in “obtaining compound is replaced with corresponding isotopes of the atoms. For intended to include purchasing, synthesizing or otherwise example, an isotopic derivative of a compound containing a 30 acquiring the compound. carbon atom (C) would be one in which the carbonatom of The term “optical isomers' as used herein includes mol the compound is replaced with the C' isotope. ecules, also known as chiral molecules, that are exact non The invention provides a number of targets that are useful Superimposable mirror images of one another. for the development of highly specific drugs to reduce fertility The phrases “parenteral administration” and “adminis in a male subject. In addition, the methods of the invention 35 tered parenterally as used herein means modes of adminis provide a facile means to identify other contraceptive thera tration other than enteral and topical administration, usually pies that are safe for use in male Subjects. In addition, the by injection, and includes, without limitation, intravenous, methods of the invention provide a route for analyzing virtu intramuscular, intraarterial, intrathecal, intracapsular, ally any number of compounds for effects on a disease intraorbital, intracardiac, intradermal, intraperitoneal, tran described herein with high-volume throughput, high sensitiv 40 stracheal, Subcutaneous, Subcuticular, intraarticulare, Sub ity, and low complexity. capsular, Subarachnoid, intraspinal and intrasternal injection By "fitting is meant determining by automatic, or semi and infusion. automatic means, interactions between one or more atoms of "Pharmaceutically acceptable” refers to approved or an agent molecule and one or more atoms or binding sites of approvable by a regulatory agency of the Federal or a state a BET family member (e.g., a bromodomain of BRD2, 45 government or listed in the U.S. Pharmacopeia or other gen BRD3, BRD4 and BRDT), and determining the extent to erally recognized pharmacopeia for use in animals, including which such interactions are stable. Various computer-based humans. methods for fitting are described further herein. "Pharmaceutically acceptable excipient, carrier or adju By "fragment' is meant a portion of a polypeptide or vant” refers to an excipient, carrier or adjuvant that can be nucleic acid molecule. This portion contains, preferably, at 50 administered to a subject, together with an agent, e.g., any of least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the compounds described herein, and which does not destroy the entire length of the reference nucleic acid molecule or the pharmacological activity thereof and is nontoxic when polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, administered in doses sufficient to deliver a therapeutic 70, 80,90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or amount of the agent. 1000 nucleotides or amino acids. 55 The terms “polycyclyl or “polycyclic radical refer to the “Hybridization” means hydrogen bonding, which may be radical of two or more cyclic rings (e.g., cycloalkyls, Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in bonding, between complementary nucleobases. For example, which two or more carbons are common to two adjoining adenine and thymine are complementary nucleobases that rings, e.g., the rings are “fused rings'. Rings that are joined pair through the formation of hydrogen bonds. 60 through non-adjacentatoms are termed “bridged rings. Each By "isolated polynucleotide' is meant a nucleic acid (e.g., of the rings of the polycycle can be substituted with such a DNA) that is free of the genes which, in the naturally Substituents as described above, as for example, halogen, occurring genome of the organism from which the nucleic hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbo acid molecule of the invention is derived, flank the gene. The nyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, term therefore includes, for example, a recombinant DNA 65 alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, that is incorporated into a vector; into an autonomously rep phosphate, phosphonato, phosphinato, cyano, amino (includ licating plasmid or virus; or into the genomic DNA of a ing alkyl amino, dialkylamino, arylamino, diarylamino, and US 9,301.962 B2 13 14 alkylarylamino), acylamino (including alkylcarbonylamino, example, a segment of a full-length cDNA or gene sequence, arylcarbonylamino, carbamoyl and ureido), amidino, imino, or the complete cDNA or gene sequence. For polypeptides, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sul the length of the reference polypeptide sequence will gener fonato, Sulfamoyl, Sulfonamido, nitro, trifluoromethyl, ally be at least about 16 amino acids, preferably at least about cyano, azido, heterocyclyl, alkyl, alkylaryl, or an aromatic or 20 amino acids, more preferably at least about 25 amino heteroaromatic moiety. acids, and even more preferably about 35 amino acids, about The term “polymorph” as used herein, refers to solid crys 50 amino acids, or about 100 amino acids. For nucleic acids, talline forms of a compound of the present invention or com the length of the reference nucleic acid sequence will gener plex thereof. Different polymorphs of the same compound ally be at least about 50 nucleotides, preferably at least about can exhibit different physical, chemical and/or spectroscopic 10 properties. Different physical properties include, but are not 60 nucleotides, more preferably at least about 75 nucleotides, limited to stability (e.g., to heat or light), compressibility and and even more preferably about 100 nucleotides or about 300 density (important in formulation and product manufactur nucleotides or any integer thereabout or therebetween. ing), and dissolution rates (which can affect bioavailability). By “specifically binds” is meant a compound or antibody Differences in Stability can result from changes in chemical 15 that recognizes and binds a polypeptide of the invention, but reactivity (e.g., differential oxidation, such that a dosage form which does not substantially recognize and bind other mol discolors more rapidly when comprised of one polymorph ecules in a sample, for example, a biological sample, which than when comprised of another polymorph) or mechanical naturally includes a polypeptide of the invention. characteristics (e.g., tablets crumble on storage as a kineti Nucleic acid molecules useful in the methods of the inven cally favored polymorph converts to thermodynamically tion include any nucleic acid molecule that encodes a more stable polymorph) or both (e.g., tablets of one poly polypeptide of the invention or a fragment thereof. Such morph are more Susceptible to breakdown at high humidity). nucleic acid molecules need not be 100% identical with an Different physical properties of polymorphs can affect their endogenous nucleic acid sequence, but will typically exhibit processing. substantial identity. Polynucleotides having “substantial The term “prodrug includes compounds with moieties 25 identity” to an endogenous sequence are typically capable of which can be metabolized in vivo. Generally, the prodrugs are hybridizing with at least one strand of a double-stranded metabolized in vivo by esterases or by other mechanisms to nucleic acid molecule. Nucleic acid molecules useful in the active drugs. Examples of prodrugs and their uses are well methods of the invention include any nucleic acid molecule known in the art (See, e.g., Berge et al. (1977) “‘Pharmaceu that encodes a polypeptide of the invention or a fragment tical Salts'. J. Pharm. Sci. 66:1-19). The prodrugs can be 30 thereof. Such nucleic acid molecules need not be 100% iden prepared in situ during the final isolation and purification of tical with an endogenous nucleic acid sequence, but will the compounds, or by separately reacting the purified com typically exhibit substantial identity. Polynucleotides having pound in its free acid form or hydroxyl with a suitable esteri “Substantial identity” to an endogenous sequence are typi fying agent. Hydroxyl groups can be converted into esters via cally capable of hybridizing with at least one strand of a treatment with a carboxylic acid. Examples of prodrug moi 35 double-stranded nucleic acid molecule. eties include substituted and unsubstituted, branch or By “hybridize' is meant pair to form a double-stranded unbranched lower alkyl ester moieties, (e.g., propionoic acid molecule between complementary polynucleotide sequences esters), lower alkenyl esters, di-lower alkyl-amino lower (e.g., a gene described herein), or portions thereof, under alkyl esters (e.g., dimethylaminoethyl ester), acylamino various conditions of stringency. (See, e.g., Wahl, G. M. and lower alkyl esters (e.g., acetyloxymethyl ester), acyloxy 40 S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters R. (1987) Methods Enzymol. 152:507). For example, strin (phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester), gent salt concentration will ordinarily be less than about 750 Substituted (e.g., with methyl, halo, or methoxy Substituents) mM NaCl and 75 mM trisodium citrate, preferably less than aryland aryl-lower alkyl esters, amides, lower-alkyl amides, about 500 mMNaCl and 50 mM trisodium citrate, and more di-lower alkyl amides, and hydroxy amides. Preferred pro 45 preferably less than about 250 mM NaCl and 25 mM triso drug moieties are propionoic acid esters and acyl esters. Pro dium citrate. Low Stringency hybridization can be obtained in drugs which are converted to active forms through other the absence of organic solvent, e.g., formamide, while high mechanisms in Vivo are also included. stringency hybridization can be obtained in the presence of at Furthermore the indication of stereochemistry across a least about 35% formamide, and more preferably at least carbon-carbon double bond is also opposite from the general 50 about 50% formamide. Stringent temperature conditions will chemical field in that “Z” refers to what is often referred to as ordinarily include temperatures of at least about 30°C., more a “cis” (same side) conformation whereas “E” refers to what preferably of at least about 37°C., and most preferably of at is often referred to as a “trans' (opposite side) conformation. least about 42° C. Varying additional parameters, such as Both configurations, cis/trans and/or Z/E are encompassed by hybridization time, the concentration of detergent, e.g., the compounds of the present invention. 55 sodium dodecyl sulfate (SDS), and the inclusion or exclusion With respect to the nomenclature of a chiral center, the of carrier DNA, are well-known to those skilled in the art. terms “d' and “1” configuration are as defined by the IUPAC Various levels of stringency are accomplished by combining Recommendations. As to the use of the terms, diastereomer, these various conditions as needed. In a preferred: embodi racemate, epimer and enantiomer, these will be used in their ment, hybridization will occur at 30°C. in 750 mM. NaCl, 75 normal context to describe the Stereochemistry of prepara 60 mM trisodium citrate, and 1% SDS. In a more preferred tions. embodiment, hybridization will occur at 37° C. in 500 mM By “reduces is meant a negative alteration of at least 10%, NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, 25%, 50%, 75%, or 100%. and 100 g/ml denatured salmon sperm DNA (ssDNA). In a By “reference' is meant a standard or control condition. most preferred embodiment, hybridization will occur at 42 A “reference sequence' is a defined sequence used as a 65 C. in 250 mM. NaCl, 25 mM trisodium citrate, 1% SDS, 50% basis for sequence comparison. A reference sequence may be formamide, and 200 g/ml ssDNA. Useful variations on these a Subset of or the entirety of a specified sequence; for conditions will be readily apparent to those skilled in the art. US 9,301.962 B2 15 16 For most applications, washing steps that follow hybrid the length of the reference polypeptide sequence will gener ization will also vary in Stringency. Wash Stringency condi ally be at least about 16 amino acids, preferably at least about tions can be defined by Salt concentration and by temperature. 20 amino acids, more preferably at least about 25 amino As above, wash stringency can be increased by decreasing acids, and even more preferably about 35 amino acids, about salt concentration or by increasing temperature. For example, 50 amino acids, or about 100 amino acids. For nucleic acids, stringent salt concentration for the wash steps will preferably the length of the reference nucleic acid sequence will gener be less than about 30 mMNaCl and 3 mM trisodium citrate, ally be at least about 50 nucleotides, preferably at least about and most preferably less than about 15 mMNaCl and 1.5 mM 60 nucleotides, more preferably at least about 75 nucleotides, trisodium citrate. Stringent temperature conditions for the and even more preferably about 100 nucleotides or about 300 wash steps will ordinarily include a temperature of at least 10 nucleotides or any integer thereabout or therebetween. about 25°C., more preferably of at least about 42°C., and By "subject' is meanta mammal, including, but not limited even more preferably of at least about 68°C. In a preferred to, a human or non-human mammal. Such as a bovine, equine, embodiment, wash steps will occur at 25°C. in 30 mMNaCl, canine, Ovine, or feline. 3 mM trisodium citrate, and 0.1% SDS. In a more preferred By “specifically binds” is meant a compound or antibody embodiment, wash steps will occur at 42 C in 15 mM NaCl, 15 that recognizes and binds a polypeptide of the invention, but 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred which does not substantially recognize and bind other mol embodiment, wash steps will occur at 68°C. in 15 mMNaCl, ecules in a sample, for example, a biological sample, which 1.5 mM trisodium citrate, and 0.1% SDS. Additional varia naturally includes a polypeptide of the invention. tions on these conditions will be readily apparent to those The term “sulfhydryl' or “thiol” means -SH. skilled in the art. Hybridization techniques are well-known to As used herein, the term “tautomers' refers to isomers of those skilled in the art and are described, for example, in organic molecules that readily interconvert by tautomeriza Benton and Davis (Science 196:180, 1977); Grunstein and tion, in which a hydrogen atom or proton migrates in the Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); reaction, accompanied in Some occasions by a Switch of a Ausubel et al. (Current Protocols in Molecular Biology, single bond and an adjacent double bond. Wiley Interscience, New York, 2001); Berger and Kimmel 25 As used herein, the terms “treat,” “treating.” “treatment.” (Guide to Molecular Cloning Techniques, 1987, Academic and the like refer to reducing orameliorating a disorder and/or Press, New York); and Sambrook et al., Molecular Cloning: A symptoms associated therewith. By “ameliorate' is meant Laboratory Manual, Cold Spring Harbor Laboratory Press, decrease, Suppress, attenuate, diminish, arrest, or stabilize the New York. development or progression of a disease. It will be appreci By “substantially identical' is meant a polypeptide or 30 ated that, although not precluded, treating a disorder or con nucleic acid molecule exhibiting at least 85% identity to a dition does not require that the disorder, condition or symp reference amino acid sequence (for example, any one of the toms associated therewith be completely eliminated. amino acid sequences described herein) or nucleic acid As used herein, the terms “prevent,” “preventing.” “preven sequence (for example, any one of the nucleic acid sequences tion.” “prophylactic treatment” and the like refer to reducing described herein). Preferably, such a sequence is at least 85%, 35 the probability of developing a disorder or condition in a 90%. 95%, 99% or even 100% identical at the amino acid subject, who does not have, but is at risk of or susceptible to level or nucleic acid to the sequence used for comparison. developing a disorder or condition. Sequence identity is typically measured using sequence “An effective amount” refers to an amount of a compound, analysis Software (for example, Sequence Analysis Software which confers a contraceptive effect on the treated subject. Package of the Genetics Computer Group, University of Wis 40 The effect may be objective (i.e., measurable by some test or consin Biotechnology Center, 1710 University Avenue, marker) or subjective (i.e., Subject gives an indication of or Madison, Wis. 53705, BLAST, BESTFIT, GAP or PILEUP/ feels an effect). An effective amount of a compound described PRETTYBOX programs). Such software matches identical herein may range from about 1 mg/Kg to about 5000 mg/Kg or similar sequences by assigning degrees of homology to body weight. Effective doses will also vary depending on various Substitutions, deletions, and/or other modifications. 45 route of administration, as well as the possibility of co-usage Conservative substitutions typically include substitutions with other agents. In embodiments of the present evention, within the following groups: glycine, alanine; Valine, isoleu “an effective amount of an agent or composition is an cine, leucine; aspartic acid, glutamic acid, asparagine, amount Sufficient to effect contraception. glutamine; serine, threonine; lysine, arginine; and phenylala Ranges provided herein are understood to be shorthand for nine, tyrosine. In an exemplary approach to determining the 50 all of the values within the range. For example, a range of 1 to degree of identity, a BLAST program may be used, with a 50 is understood to include any number, combination of num probability score betweene.sup.-3 and e.sup.-100 indicating bers, or Sub-range from the group consisting of 1, 2, 3, 4, 5, 6, a closely related sequence. 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, By “reduces” or “increases” is meant a negative or positive 25, 26, 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37,38, 39, 40, 41, alteration, respectively, of at least about 10%, 25%, 50%, 55 42, 43, 44, 45, 46, 47, 48, 49, or 50. 75%, or 100% relative to a reference. Unless specifically stated or obvious from context, as used By "root mean square deviation' is meant the square root herein, the term 'or' is understood to be inclusive. Unless of the arithmetic mean of the squares of the deviations from specifically stated or obvious from context, as used herein, the the mean. terms “a”, “an', and “the are understood to be singular or By “reducing cell survival' is meant to inhibit the viability 60 plural. of a cell or to induce cell death relative to a reference cell. The term “including is used herein to mean, and is used By “reference' is meant a standard or control condition. interchangeably with, the phrase “including but not limited A “reference sequence' is a defined sequence used as a to. basis for sequence comparison. A reference sequence may be Unless specifically stated or obvious from context, as used a Subset of or the entirety of a specified sequence; for 65 herein, the term “about is understood as within a range of example, a segment of a full-length cDNA or gene sequence, normal tolerance in the art, for example within 2 standard or the complete cDNA or gene sequence. For polypeptides, deviations of the mean. About can be understood as within US 9,301.962 B2 17 18 10%, 9%, 8%, 7%, 6%. 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, weeks of age. FIG. 6B includes a graph showing the in vitro 0.05%, or 0.01% of the stated value. Unless otherwise clear developmental potential of oocytes obtained from SuperoVu from context, all numerical values provided herein are modi lated females mated to males treated for 5 weeks with a fied by the term about. control or JQ1. All data represent the meaniSEM, and are The recitation of a listing of chemical groups in any defi 5 annotated with P-values as obtained from a two-tailed t-test (* nition of a variable herein includes definitions of that variable indicates significant at PK0.05). as any single group or combination of listed groups. The FIGS. 7A-7C show the molecular analysis of the testes of recitation of an embodiment for a variable or aspect herein mice treated with JQ1 or control. FIG. 7A includes a graph includes that embodiment as any single embodiment or in showing the quantitative RT-PCR results for males treated combination with any other embodiments orportions thereof. 10 from 6-12 weeks of age with JQ1 or a control solution. The Any compounds, compositions, or methods provided mouse genes tested were Plzf (promyelocytic leukemia zinc herein can be combined with one or more of any of the other finger or Zbtb16), Stra8 (stimulated by retinoic acid gene 8), compounds, compositions, and methods provided herein. Brdt (bromodomain, testis-specific), Ccnal (cyclin A1). DESCRIPTION OF THE DRAWINGS 15 Hist1 hlt (histone cluster 1, histone 1, testis-specific), Papolb (poly (A) polymerase beta or Tpap), Klf17 (Kruppel-like FIG. 1 is a sequence comparison of human BRDT(1) with factor 17 or Zfp393), and Prm 1 (protamine 1). Data represent human BRD4(1) and mouse BRDT(1). Protein sequence the meantSEM, and are annotated with P-values as obtained alignment reveals a high degree of sequence identity between from a two-tailed t-test (* indicates significant at PK0.05; the homologous and orthologous domains. Identical (red) and P-value for Prm1 is 0.06). FIGS. 7B and 7C include immu similar (yellow) residues are highlighted. Depicted above the nohistochemical staining images of TNP2 in control-treated residue sequences are schematic representations of major (FIG. 7B) and JQ1-treated (FIG. 7C) testes. helical elements. Contacts between (+)-JQ1 and BRDT(1) FIGS. 8A-8C show the effect of JQ1 on sperm count and are depicted with a black star. The conserved asparagine testicular mass. In a repeat study, C57B6 mice were treated mediating acetyl-lysine recognition is depicted with a blue 25 with JO1 (50 mpkx8 weeks). FIG. 8A includes a graph show Star. ing sperm count in the test mice. FIG. 8B includes a graph FIGS. 2A-2C show BRDT inhibition by (+)-JQ1. FIG. 2A showing testes weight in the test mice. FIG. 8C includes is the structure of the active (+)-JQ1 enantiomer. FIG. 2B is a phase contrast images of sperm from test mice. plot showing the competitive inhibition of BRDT binding to FIGS. 9A-9C show that the effects of JQ1 are reversed synthetic biotinylated H4Kac4 by (+)-JQ1 (IC50: 11 nM) 30 using a proximity detection assay. FIG. 2C includes a graph upon removal. FIG. 9A includes a graph showing sperm showing the results of the assay with 500 nM of the indicated motility levels in test mice two months and four months after compound. Error bars show standard deviation. termination of JQ1 treatment. FIG.9B includes a graph show FIG.3 is a MAFFT alignment of human BRDT and human ing testes weight in test mice two months and four months BRD4. 35 after termination of JQ1 treatment. FIG.9C includes a graph FIG. 4 is a MAFFT alignment of human BRDT and mouse showing the sperm counts in test mice two months and four BRDT. months after termination of JQ1 treatment. FIGS.5A-5H show gross and histological analysis oftestes from mice treated with JQ1 or vehicle control. FIG. 5A DETAILED DESCRIPTION OF THE INVENTION includes an image providing a gross analysis of testes from 40 9-week old mice injected with control or JQ1. FIG. 5B This invention is based, at least in part, on the discovery includes a graphical representation of testes weights (mg) that a small-molecule inhibitor (JO1) of the bromodomain from mice treated with control or JQ1 for 3-6 weeks, 6-9 and extra-terminal (BET) subfamily of epigenetic reader pro weeks, or 6-12 weeks of age. Data represent the teins is essential for chromatin remodeling during spermio meanistandard error of the mean (SEM), and are annotated 45 genesis. Biochemical analysis confirms that occupancy of the with P-values as obtained from a two-tailed t-test (* indicates BRDT acetyl-lysine binding pocket by JQ1 prevents recog significant at P<0.05). FIGS. 5C-5F include histological nition of acetylated histone H4. The invention is also based on stains showing the histology of testes of 6-week old mice the discovery that treatment of mice with JQ1 reduced the treated with (FIG. 5C) control or (FIG. 5D) JQ1 from 3-6 number and motility of spermatozoa, as well as testis size. weeks of age, and 12-week old mice treated with (FIG. 5E) 50 Although JQ1-treated males mate normally, inhibitory effects control or (FIG.5F) JQ1 from 6-12 weeks of age. Intertubular of JQ1 evident at the spermatocyte stage cause a dramatic islands of Leydig cells are depicted with arrows in FIGS. decrease in fertilized oocytes and a reversible contraceptive 5C-5F. Sertolicell vacuolization (V) is highlighted in several effect in males. Accordingly, the present invention is directed tubules in FIG. 5F. FIGS. 5G and 5H include histological to a novel type of male contraceptive that can cross the blood: stains showing the histology of the epididymides from males 55 testis boundary and inhibits bromodomain activity during treated with (FIG.5G) control or (FIG. 5H) JQ1 from 6-12 Spermatogenesis. weeks of age. Fewer spermatozoa and multiple large nucle Bromodomain-containing Proteins ated cells (black arrow) are observed in the epididymal lumen Gene regulation is fundamentally governed by reversible, of the JO1-treated mice (FIG. 5H) compared to the control non-covalent assembly of macromolecules. Signal transduc epididymal lumen (FIG.5G), which is densely packed with 60 tion to RNA polymerase requires higher-ordered protein mature spermatozoa. FIGS. 5C-5H were photographed at the complexes, spatially regulated by assembly factors capable of same magnification. interpreting the post-translational modification states of chro FIGS. 6A and 6B show epididymal sperm counts and fer matin. Epigenetic readers are structurally diverse proteins tilization potential. FIG. 6A is a graphical representation of each possessing one or more evolutionarily conserved effec the sperm counts obtained from the entire epididymides of 65 tor modules, which recognize covalent modifications of his males treated with JO1 or control from 6-9 weeks of age or the tone proteins or DNA. The e-N-acetylation of lysine residues tail (cauda) of the epididymis of males treated from 6-12 (Kac) on histone tails is associated with an open chromatin US 9,301.962 B2 19 20 architecture and transcriptional activation. Context-specific wherein molecular recognition of acetyl-lysine is principally medi X is N or CR; ated by bromodomains. Rs is H. alkyl, cycloalkyl, heterocycloalkyl, aryl, or het Bromodomain-containing proteins are of Substantial bio eroaryl, each of which is optionally substituted; logical interest, as components of transcription factor com 5 R is H. alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, plexes (TAF1, PCAF, GcnS and CBP) and determinants of haloalkyl, hydroxy, alkoxy, or —COO—R, each of epigenetic memory'. There are 41 human proteins containing which is optionally substituted; a total of 57 diverse bromodomains. Despite large sequence ring A is aryl or heteroaryl; variations, all bromodomains share a conserved fold compris each R is independently alkyl, cycloalkyl, heterocy 10 cloalkyl, aryl, or heteroaryl, each of which is option ing a left-handed bundle of four alpha helices (C. C., C. ally substituted; or any two R, together with the atoms O), linked by diverse loop regions (ZA and BC loops) that to which each is attached, can form a fused aryl or determine substrate specificity. Co-crystal structures with heteroaryl group; peptidic Substrates showed that the acetyl-lysine is recog R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; nized by a central hydrophobic cavity and is anchored by a 15 each of which is optionally substituted; hydrogen bond with an asparagine residue present in most R is —(CH)-L, in which n is 0-3 and L is H. —COO– bromodomains. The bromodomain and extra-terminal R. —CO—Rs. -CO—N(RR). —S(O) R. (BET)-family (BRD2, BRD3, BRD4 and BRDT) shares a —S(O), N(RR), N(RR), N(R)C(O)R option common domain architecture comprising two N-terminal ally substituted aryl, or optionally substituted het bromodomains that exhibit high level of sequence conserva eroaryl; tion, and a more divergent C-terminal recruitment domain. R is H. D (deuterium), halogen, or optionally substituted The invention features compositions and methods that are alkyl: useful for inhibiting human bromodomain proteins. each R is independently selected from the group consist Compounds of the Invention ing of The invention provides compounds (e.g., JO1 and com 25 (i) H, aryl, substituted aryl, heteroaryl, or substituted pounds of formulas delineated herein) that bind in the binding heteroaryl; pocket of the apocrystal structure of the first bromodomain of (ii) heterocycloalkyl or substituted heterocycloalkyl: a BET family member (e.g., BRDT, BRD2, BRD3, BRD4). (iii)—C-C alkyl, -C-C alkenyl or—C-C alkynyl, Without wishing to be bound by theory, these compounds are each containing 0, 1, 2, or 3 heteroatoms selected 30 from O, S, or N. —C-C cycloalkyl, substituted particularly effective in reducing male fertility. In one —C-C cycloalkyl, -C-C cycloalkenyl, or Sub approach, compounds useful for reducing male fertility are stituted—C-C cycloalkenyl, each of which may be selected using a molecular docking program to identify com optionally substituted; and pounds that are expected to bind to a bromodomain structural (iv) NH, N=CRR: binding pocket. In certain embodiments, a compound of the 35 each R is independently H, alkyl, alkyl, cycloalkyl, invention can prevent, inhibit, or disrupt, or reduce by at least heterocycloalkyl, aryl, or heteroaryl, each of which is 10%, 25%, 50%, 75%, or 100% the biological activity of a optionally substituted; BET family member (e.g., BRD2, BRD3, BRD4, BRDT) or R and Rare taken together with the nitrogenatom to and/or disrupt the subcellular localization of such proteins, which they are attached to form a 4-10-membered e.g., by binding to a binding site in a bromodomain apo 40 r1ng, binding pocket. R is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocy In certain embodiments, a compound of the invention is a cloalkyl, aryl, or heteroaryl, each of which is option Small molecule having a molecular weight less than about ally Substituted; or R and R are taken together with 1000 daltons, less than 800, less than 600, less than 500, less the carbon atom to which they are attached to form a than 400, or less than about 300 daltons. Examples of com 45 4-10-membered ring: pounds of the invention include JO1 and other compounds m is 0, 1, 2, or 3: that bind the binding pocket of the apocrystal structure of the provided that first bromodomain of a BET family member (e.g., BRD4 (a) ifring A is thienyl, X is N, R is phenyl or substituted (hereafter referred to as BRD4(1): PDB ID 20SS). JQ1 is a phenyl, R is H. R. is methyl, and R is —(CH)-L. novel thieno-triazolo-1,4-diazepine. The invention further 50 in which n is 1 and L is —CO N(RR), then R and provides pharmaceutically acceptable salts of Such com Ra are not taken together with the nitrogen atom to pounds. which they are attached to form a morpholino ring; In one aspect, the compound is a compound of Formula I: (b) ifring A is thienyl, X is N, R is substituted phenyl, R. is H. R. is methyl, and R is —(CH),L, in which in 55 is 1 and L is —CO—N(RR), and one of R and R is H, then the other of RandR is not methyl, hydroxy R (I) ethyl, alkoxy, phenyl, Substituted phenyl, pyridyl or substituted pyridyl; and
w R (c) ifring A is thienyl, X is N, R is substituted phenyl, R. 60 is H. R. is methyl, and R is —(CH2)-L, in which in (R)-- A || is 1 and L is COO R, then R is not methyl or ethyl: N or a salt, solvate or hydrate thereof. N / RB X In certain embodiments, R is aryl or heteroaryl, each of 65 which is optionally substituted. In certain embodiments, L is H. —COO R. —CO N (RR). —S(O) R. —S(O), N(RR), N(RR), N(R) US 9,301.962 B2 21 22 C(O)R or optionally substituted aryl. In certain embodi m is 0, 1, 2, or 3: ments, each R is independently selected from the group provided that if R', is —COO RX is N, R is substituted consisting of H. —C-Cs alkyl, containing 0, 1, 2, or 3 phenyl, and R is methyl, then R is not methyl or ethyl, heteroatoms selected from O, S, or N; or NH, N=CRR. or a salt, solvate or hydrate thereof. In certain embodiments, R is H. D., halogen or methyl. In certain embodiments, R is aryl or heteroaryl, each of In certain embodiments, R is alkyl, hydroxyalkyl, which is optionally substituted. In certain embodiments, R is haloalkyl, or alkoxy; each of which is optionally substituted. phenyl or pyridyl, each of which is optionally substituted. In In certain embodiments, R is methyl, ethyl, hydroxy certain embodiments, R is p-Cl-phenyl, o-C1-phenyl, m-Cl methyl, methoxymethyl, trifluoromethyl, COOH, COOMe, phenyl, p-F-phenyl, o-F-phenyl, m-F-phenyl or pyridinyl. COOEt, or COOCHOC(O)CH. 10 In certain embodiments, R', is —COO—R, optionally In certain embodiments, ring A is a 5 or 6-membered aryl substituted aryl, or optionally substituted heteroaryl; and R. or heteroaryl. In certain embodiments, ring A is thiofuranyl. is —C-C alkyl, which contains 0, 1, 2, or 3 heteroatoms phenyl, naphthyl, biphenyl, tetrahydronaphthyl, indanyl. selected from O, S, or N, and which may be optionally sub pyridyl, furanyl, indolyl pyrimidinyl, pyridizinyl, pyrazinyl, stituted. In certain embodiments, R', is —COO RandR 15 is methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, or t-butyl, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, or R', is H or optionally substituted phenyl. quinolinyl, pyrrolyl, pyrazolyl, or 5.6.7.8-tetrahydroiso In certain embodiments, R is methyl, ethyl, hydroxy quinolinyl. methyl, methoxymethyl, trifluoromethyl, COOH, COOMe, In certain embodiments, ring A is phenyl or thienyl. COOEt, COOCHOC(O)CH, In certain embodiments, m is 1 or 2, and at least one In certain embodiments, R is methyl, ethyl, hydroxy occurrence of R is methyl. methyl, methoxymethyl, trifluoromethyl, COOH, COOMe, In certain embodiments, each R is independently H, an COOEt, or COOCHOC(O)CH, optionally substituted alkyl, or any two R, together with the In certain embodiments, each R is independently an atoms to which each is attached, can form an aryl. optionally substituted alkyl, or any two R together with the In another aspect, the compound is a compound of Formula 25 atoms to which each is attached, can form a fused aryl. II: In certain embodiments, each R is methyl. In another aspect, the compound is a compound of formula III: (II) R 30 SN R (III) (RA) {a N S N lsN \/ R X wherein 40 X is N or CRs: Rs is H. alkyl, cycloalkyl, heterocycloalkyl, aryl, or het wherein eroaryl, each of which is optionally substituted; X is N or CRs: R is H. alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, Rs is H. alkyl, cycloalkyl, heterocycloalkyl, aryl, or het haloalkyl, hydroxy, alkoxy, or —COO R, each of 45 eroaryl, each of which is optionally substituted; which is optionally substituted; R is H. alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, each R is independently alkyl, cycloalkyl, heterocy haloalkyl, hydroxy, alkoxy, or —COO R, each of cloalkyl, aryl, or heteroaryl, each of which is optionally which is optionally substituted; substituted; or any two R together with the atoms to ring A is aryl or heteroaryl; which each is attached, can form a fused aryl or het 50 each R is independently alkyl, cycloalkyl, heterocy eroaryl group; cloalkyl, aryl, or heteroaryl, each of which is option ally substituted; or any two R, together with the atoms R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, to which each is attached, can form a fused aryl or each of which is optionally substituted; heteroaryl group; R", is H. —COO R. —CO R, optionally substituted 55 R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, aryl, or optionally substituted heteroaryl; each of which is optionally substituted; each R is independently selected from the group con each R is independently selected from the group consist sisting of: ing of (i) H, aryl, substituted aryl, heteroaryl, substituted het (i) H, aryl, substituted aryl, heteroaryl, or substituted eroaryl; 60 heteroaryl; (ii) heterocycloalkyl or substituted heterocycloalkyl: (ii) heterocycloalkyl or substituted heterocycloalkyl: (iii)—C-C alkyl, -C-Cs alkenylor-C-Cs alkynyl, (iii)—C-Cs alkyl, -C-Cs alkenylor-C-Cs alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N. —C-C cycloalkyl, substituted from O, S, or N. —C-C cycloalkyl, substituted —C-C cycloalkyl; —C-C cycloalkenyl, or Sub 65 —C-C cycloalkyl, -C-C cycloalkenyl, or Sub stituted—C-C cycloalkenyl; each of which may be stituted—C-C cycloalkenyl, each of which may be optionally substituted; optionally substituted; and US 9,301.962 B2 23 24 (iv) NH, N=CRR: R is H. alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, each R is independently H, alkyl, alkyl, cycloalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R, each of heterocycloalkyl, aryl, or heteroaryl, each of which is which is optionally substituted; optionally substituted; ring A is aryl or heteroaryl; or R and Rare taken together with the nitrogenatom to each R is independently alkyl, cycloalkyl, heterocy which they are attached to form a 4-10-membered cloalkyl, aryl, or heteroaryl, each of which is option ring: ally substituted; or any two R, together with the atoms R is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocy to which each is attached, can form a fused aryl or cloalkyl, aryl, or heteroaryl, each of which is option 10 heteroaryl group; ally Substituted; or R and R are taken together with R is —(CH),-L, in which n is 0-3 and L is H. —COO— the carbon atom to which they are attached to form a R. —CO—Rs. -CO—N(RR). —S(O) R. 4-10-membered ring: —S(O), N(RR), N(RR), N(R)C(O)R option m is 0, 1, 2, or 3: 15 ally substituted aryl, or optionally substituted het provided that: eroaryl; (a) ifring A is thienyl, X is N, R is phenyl or substituted R is H. D., halogen, or optionally substituted alkyl, phenyl, R is methyl, then R and Ra are not taken each R is independently selected from the group consist together with the nitrogen atom to which they are ing of attached to form a morpholino ring; and (i) H, aryl, substituted aryl, heteroaryl, or substituted (b) ifring A is thienyl, X is N, R is substituted phenyl, R. heteroaryl; is H. R. is methyl, and one of R and R is H, then the (ii) heterocycloalkyl or substituted heterocycloalkyl: other of R and R is not methyl, hydroxyethyl, (iii)—C-Cs alkyl, -C-Cs alkenylor-C-Cs alkynyl, alkoxy, phenyl, Substituted phenyl, pyridyl or Substi 25 each containing 0, 1, 2, or 3 heteroatoms selected tuted pyridyl; and from O, S, or N. —C-C cycloalkyl, substituted or a salt, solvate or hydrate thereof. —C-C cycloalkyl, -C-C cycloalkenyl, or Sub In certain embodiments, R is aryl or heteroaryl, each of stituted—C-C cycloalkenyl, each of which may be which is optionally substituted. In certain embodiments, R is 30 optionally substituted; and phenyl or pyridyl, each of which is optionally substituted. (iv) NH, N=CRR: In certain embodiments, R is p-Cl-phenyl, o-Cl-phenyl, each R is independently H, alkyl, alkyl, cycloalkyl, m-Cl-phenyl, p-F-phenyl, o-F-phenyl, m-F-phenyl or pyridi heterocycloalkyl, aryl, or heteroaryl, each of which is nyl. In certain embodiments, R is H, NH, or N=CRR. optionally substituted; In certain embodiments, each R is independently H, alkyl, 35 or R and Rare taken together with the nitrogenatom to cycloalkyl, heterocycloalkyl, aryl, heteroaryl; each of which which they are attached to form a 4-10-membered is optionally substituted. ring: In certain embodiments, R is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of 40 R is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocy which is optionally substituted. cloalkyl, aryl, or heteroaryl, each of which is option In another aspect, the compound is a compound of formula ally substituted; or R and Rare taken together with IV: the carbon atom to which they are attached to form a 4-10-membered ring: 45 m is 0, 1, 2, or 3: provided that (a) ifring A is thienyl, X is N, R is H. R. is methyl, and
(IV) R is —(CH),-L, in which n is 0 and L is —CO N (RR), then R and Rare not taken together with the 50 nitrogen atom to which they are attached to form a morpholino ring: (b) ifring A is thienyl, X is N, R is H. R. is methyl, and R is —(CH),-L, in which n is 0 and L is —CO N 55 (RR), and one of R and R is H, then the other of Rs. and R is not methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl; and (c) ifring A is thienyl, X is N, R2 is H. R. is methyl, and R is —(CH),-L, in which n is 0 and L is —COO– 60 R, then R is not methyl or ethyl; or a salt, solvate or hydrate thereof. In certain embodiments, R is —(CH)-L, in which n is wherein 0-3 and L is —COO R, optionally substituted aryl, or X is N or CRs: 65 optionally substituted heteroaryl; and R is —C-C alkyl, Rs is H. alkyl, cycloalkyl, heterocycloalkyl, aryl, or het which contains 0, 1, 2, or 3 heteroatoms selected from O, S, or eroaryl, each of which is optionally substituted; N, and which may be optionally substituted. In certain US 9,301.962 B2 25 26 embodiments, n is 1 or 2 and L is alkyl or —COO R, and In another aspect, the compound is a compound repre R is methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, ort-bu- sented by the formula: tyl; or n is 1 or 2 and L is H or optionally substituted phenyl. In certain embodiments, R is H or methyl. 5 In certain embodiments, R is methyl, ethyl, hydroxy methyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, COOCHOC(O)CH. In certain embodiments, ring A is phenyl, naphthyl, biphe- 10 nyl, tetrahydronaphthyl, indanyl, pyridyl, furanyl, indolyl, pyrimidinyl, pyridizinyl, pyrazinyl, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, or 5,6,7,8-tetrahydroisoquinolinyl. 15 W \ In certain embodiments, each R is independently an N1 N. Ó optionally substituted alkyl, or any two R, together with the S W atoms to which each is attached, can form an aryl. era. The methods of the invention also relate to compounds of 20 Formulae V-XXII, and to any compound described herein. In another aspect, the compound is a compound repre- 2N sented by the formula: 25 N-/
C 30 O Y. s
S r X 40 \ () $1 N NS or a salt, solvate or hydrate thereof. N N)- Cl, or 45 \ -2 N -eN In certain embodiments, the compound is (+)-JQ1: HN
O 50
C
55
e-N etN 2 W \ - 60 W \ r rol S y=N Y. O X N1 N. O OH: 65 or a salt, solvate or hydrate thereof. or a salt, solvate or hydrate thereof. US 9,301.962 B2 27 28 In another aspect, the compound is a compound repre sented by the formula:
C OH
etN O O O
Ulr-OSS 3- O H O C O
HN NH H etN O O H W \ N-1N1 N-1 no-1S-1 N-1N1 ^-n- S S N1N 6 O y= NW
30 or a salt, solvate or hydrate thereof. -continued In another aspect, the compound is a compound repre sented by any one of the following formulae: JQ11 35 N N JQ1S F e YN O ) / Y. N N O y S N A O S N / )- 40 N III eN
45
C 50
sy\ JQIR N NH N YyN O NR 55 Y-yN O y
60
65 C C
US 9,301.962 B2 31 32 In another aspect, the compound is a compound repre- -continued sented by any one of the following formulae: 2N N N-/ C N -eN )- NH O YH, 10 etN O NH
S CN N O C y= W 15 2NYs OH N-/
C III B(OH)2: 2O N eN NH
O) MNR
eN 25 W \ N4 C S N1 N. (3 21 NN y--/ N 30 Y.N-/ N S N III or a salt, solvate or hydrate thereof. -e-N X-y N le In another aspect, the compound is a compound repre 35 O N NH sented by any one of the following structures: Y
N-y- 40 N-yN ... I I III S N-/ Y-N- )- 45
SONa 50 / 2
N N Yy2 N 55 Yy21 N N-/ N-/
65 C C US 9,301.962 B2 33 34 -continued -continued - 2N N QuS N - II y 5 eN X O 10
15 C () s II X- 2O
25
30
C
35
40
45
50 C
C 55
etN 60
S M N SN 65 C US 9,301.962 B2 35 36 -continued y)-continued
15
25
30
35
40
45
50
C
55
60 US 9,301.962 B2 37 38 -continued -continued
NH
10
15
N
25
C
30 C
or a salt, solvate or hydrate thereof. In certain embodiments, a compound of the invention can 35 be represented by one of the following structures:
40 r le N NH 45 CCy
50
C
55
60
65 C C
US 9,301.962 B2 41 42 -continued -continued 4N C ulyN-N N 4 N -e-N- // S
10 Cl C
15
N
25 C
30
35
40
45
50
55
60
65 or a salt, solvate or hydrate thereof. US 9,301.962 B2 43 44 In one embodiment, the compound is represented by the In certain embodiments, a compound of the invention can Structure: have the opposite chirality of any compound shown herein. In certain embodiments, the compound is a compound represented by Formula (V), (VI), or (VII): 5
(V)
15
C (VI) or a salt, solvate or hydrate thereof. In another embodiment, the compound is represented by the structure:
25
30
(VII)
C
or a salt, solvate or hydrate thereof. 40 In another embodiment, the compound is represented by the structure:
45 in which R, R, and R2 and R have the same meaning as in Formula (I); Y is O. N. S. or CRs, in which Rs has the same meaning as in Formula (I); n is 0 or 1; and the dashed circle in 50 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), R represents the non-carbonyl por tion of an aldehyde shown in Table A, below (i.e., for an 55 aldehyde of formula RCHO, R is the non-carbonyl portion of the aldehyde). In certain embodiments, RandR together C represent the non-carbonyl portion of a ketone shown in Table A (i.e., for a ketone of formula RC(O)R. R. and R are the or a salt, solvate or hydrate thereof. non-carbonyl portion of the ketone). US 9,301.962 B2 45 46
n
S 1 a O N N
O a e a s I I O O s S S.s O n Y in
O O I O O TZ a O
O a S- O
O i s W a YZ -\ . S o ce 2 A c==c C
O I O O
S 9- O n 4.
I O O \ a NZ
r s aY Z, Sn M
A 4 M O O
r r
US 9,301.962 B2 67 68 In one embodiment, the compound is a compound is rep -continued
resented by the formula: (X)
(VIII) 5
10
15 or a salt, solvate, or hydrate thereof. In certain embodiments, the compoundis (racemic) JO1, in (XI) certain embodiments, the compound is (+)-JQ1. In certain embodiments, the compound is a compound selected from the group consisting of (3) C 25 C
etN 30 H N / \ N NH and S N Y. O (XII) C )-ser (4)4 35
40 C e-N H N / \ N N le. S N N O 45 )-N OH, or a salt, solvate, or hydrate thereof. 50 Additional examples of compounds include compounds according to any of the follow formulae: (IX) (XIII)
55
60
65 US 9,301.962 B2 69 70 -continued (XIV) -continued (XIX)
10
(XX)
(XV) 15
25 (XXI) (XVI)
30
35
(XXII) (XVII) 40
45
50 In Formulae IX-XXII, R and R' can be, e.g., H, aryl, sub stituted aryl, heteroaryl, heteroaryl, heterocycloalkyl, —C- (XVIII) Cs alkyl, -C-C alkenyl, -C-C alkynyl, -C-C, cycloalkyl, Substituted —C-C cycloalkyl, -C-C2 55 cycloalkenyl, or Substituted —C-C cycloalkenyl, each of 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, 60 the chemical Examples provided hereinbelow provide syn thetic schemes for the preparation of the compound JO1 (as the racemate) and the enantiomers (+)-JQ1 and (-)-JQ1 (see Schemes S1 and S2). A variety of compounds of Formulae (I)-(VIII) can be prepared by analogous methods with substi 65 tution of appropriate starting materials. Also 2- and 4-pyridyl For example, starting from JO1, the analogous amine can be prepared as shown in Scheme 1, below. US 9,301.962 B2 71 72
Scheme 1
1) DPPA, NEt C - s 2) BzOH
O O NHCbz
BBr: S
1) RCHO )--SWN ) NaBHoAe,NaBH(OAc)3 N N2 NM C
N- NH NH2 R R
As shown in Scheme 1, hydrolysis of the t-butyl ester of 30 -continued JQ1 affords the carboxylic acid, which is treated with diphe nylphosphoryl azide (DPPA) and subjected to Curtius rear
rangement conditions to provide the Cbz-protected amine, which is then deprotected to yield the amine. Subsequent elaboration of the amine group, e.g., by reductive amination is yields secondary amines, which can be further alkylated to provide tertiary amines.
40
Scheme 2
45
50
-- He
55
60
65 US 9,301.962 B2 73 74 Scheme 2 shows the synthesis of further examples of the group appendages (corresponding to group R in Formula I). compounds of the invention, e.g., of Formula I, in which the Such aminodiarylketones are commercially available or can fused ring core is modified (e.g., by substitution of a different be prepared by a variety of methods, some of which are aromatic ring as Ring A in Formula I). Use of aminodiarylke 5 known in the art. tones having appropriate functionality (e.g., in place of the aminodiarylketone S2 in Scheme 51, infra) provides new Scheme 3 provides additional exemplary synthetic compounds having a variety of fused ring cores and/or aryl schemes for preparing further compounds of the invention.
Scheme 3
1) Base C -- LDA, DAMBr 2) DO, or MeI
C US 9,301.962 B2 75 76 As shown in Scheme 3, a fused bicyclic precursor (see -continued Scheme 51, infra, for synthesis of this compound) is func- \ tionalized with a moiety R (DAM-dimethylaminomethylene N protecting group) and then elaborated by reaction with a () S N hydrazide to form the tricyclic fused core. Substituent Rx can 5 be varied by selection of a suitable hydrazide. NS Additional examples of compounds of the invention ^ C (which can be prepared by the methods described herein) \ 2 / include: 10 N N Amides: NH Amides can be prepared, e.g., by preparation of a corre sponding carboxylic acidorester, followed by amidation with O an appropriate amine using standard conditions. In certain embodiments, an amide provides a two-carbon “linker with 15 a terminal terminal nitrogen-containing ring (e.g., pyridyl, piperidyl, piperazinyl, imidazolyl (including N-methyl-imi dazolyl), morpholinyl, and the like. Exemplary amide struc tures include: 2O
25
)- NS 30 N C \ 2 / si-sy\ 35 )--S$1 N N C \ 2 / N N
40 1. - (1)\=/ O
45 The use of a two-carbon linker between the amide moiety and the terminal nitrogen-containing ring is preferred. “Reverse Amides:
50
55 / N N N
60 HN
O US 9,301.962 B2 77 78 -continued -continued
C
O 10
2N. SJ 15 N position can be different
2O
C
30 C Secondary Amines:
35
45
50
55
60
65 US 9,301.962 B2 79 80 -continued -continued C B(OH)2.
ear N 2 10 / \ N S N1N yN
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 enan tiomerically enriched, i.e., has an enantiomeric excess (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 configura tion as the compound (+)-JQ1 ((S)-tert-Butyl 2-(4-(4-chlo rophenyl)-2,3,9-trimethyl-6H-thieno3.2-f 1.2.4 triazolo4. 25 3-a 1,4diazepin-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
R4 35
C 40
N H 45 in which: R", is C-C alkyl, R" is hydrogen, halogen, or C-C alkyl optionally Substi tuted with a halogen atom or a hydroxyl group; R" is a halogen atom, phenyl optionally substituted by a 50 halogen atom, C-C alkyl, C-C alkoxyy, or cyano; Boronic Acids: —NRs—(CH), R wherein Rs is a hydrogen atom or C-C alkyl, misan integer of 0-4, and R is phenyl or pyridyl optionally Substituted by a halogen atom; or —NR, CO C (CH), Rs wherein R, is a hydrogenatom or C-C alkyl. n. 55 is an integer of 0-2, and Rs is phenyl or pyridyl optionally Substituted by a halogen atom; and R" is —(CH) CO—NH-R whereina is an integer of 1-4, and Ro is C-C alkyl, C-C hydroxyalkyl, C-C, alkoxy; or phenyl or pyridyl optionally substituted by C-C, eN 60 alkyl, C-C alkoxy, amino or a hydroxyl group or —(CH), COOR wherein b is an integer of 1-4, and Rois NH C-C alkyl. N N O The term “pharmaceutically acceptable salt also refers to W a salt prepared from a compound disclosed herein (e.g., JO1, 65 a compound of Formulas I-XXII) or any other compound delineated herein, having an acidic functional group. Such as a carboxylic acid functional group, and a pharmaceutically US 9,301.962 B2 81 82 acceptable inorganic or organic base. Suitable bases include, contraceptive is a modulator of testosterone production, but are not limited to, hydroxides of alkali metals such as androgen receptor function or stability. Sodium, potassium, and lithium; hydroxides of alkaline earth Pharmaceutical Compositions metal Such as calcium and magnesium; hydroxides of other The invention features pharmaceutical compositions that metals, such as aluminum and Zinc, ammonia, and organic contain one or more of the compounds described herein, a amines, such as unsubstituted or hydroxy-Substituted mono-, derivative thereof, or a pharmaceutically acceptable salt or di-, or trialkylamines; dicyclohexylamine; tributyl amine; prodrug thereofas the active ingredient(s). The pharmaceu pyridine; N-methyl-N-ethylamine; diethylamine; triethy tical compositions contain a pharmaceutically acceptable car lamine; mono-, bis-, or tris-(2-hydroxy-lower alkyl amines), rier, excipient, or diluent, which includes any pharmaceutical Such as mono-, bis-, or tris-(2-hydroxyethyl)-amine, 2-hy 10 agent that does not itself induce the production of an immune droxy-tert-butylamine, or tris-(hydroxymethyl)methy response harmful to a subject receiving the composition, and lamine, N.N.-di-lower alkyl-N-(hydroxy lower alkyl)- which may be administered without undue toxicity. As used amines, such as N,N-dimethyl-N-(2-hydroxyethyl)-amine, or herein, the term “pharmaceutically acceptable' means being tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and approved by a regulatory agency of the Federal or a state amino acids Such as arginine, lysine, and the like. The term 15 government or listed in the U.S. Pharmacopia, European “pharmaceutically acceptable salt also refers to a salt pre Pharmacopia or other generally recognized pharmacopia for pared from a compound disclosed herein, or any other com use in mammals, and more particularly in humans. These pound delineated herein, having a basic functional group, compositions can be useful as a male contraceptive. Such as an amino functional group, and a pharmaceutically A thorough discussion of pharmaceutically acceptable car acceptable inorganic or organic acid. Suitable acids include, riers, diluents, and other excipients is presented in Reming but are not limited to, hydrogen Sulfate, citric acid, acetic ton's Pharmaceutical Sciences (17th ed., Mack Publishing acid, oxalic acid, hydrochloric acid, hydrogen bromide, Company) and Remington. The Science and Practice of hydrogen iodide, nitric acid, phosphoric acid, isonicotinic Pharmacy (21st ed., Lippincott Williams & Wilkins), which acid, lactic acid, Salicylic acid, tartaric acid, ascorbic acid, are hereby incorporated by reference. The formulation of the Succinic acid, maleic acid, besylic acid, fumaric acid, glu 25 pharmaceutical composition should suit the mode of admin conic acid, glucaronic acid, saccharic acid, formic acid, ben istration. In embodiments, the pharmaceutical composition is Zoic acid, glutamic acid, methanesulfonic acid, ethane Suitable for administration to humans, and can be sterile, Sulfonic acid, benzenesulfonic acid, and p-toluenesulfonic non-particulate and/or non-pyrogenic. acid. Pharmaceutically acceptable carriers, excipients, or dilu Methods of the Invention 30 ents include, but are not limited, to saline, buffered saline, The present invention also relates to using the novel com dextrose, water, glycerol, ethanol, Sterile isotonic aqueous pounds described herein, as well as other inhibitors of BRDT buffer, and combinations thereof. as male contraceptives. Such compounds are known in the art Wetting agents, emulsifiers and lubricants, such as Sodium and described, for example, in WO2009084693 or corre lauryl Sulfate and magnesium Stearate, as well as coloring sponding US2010286127. 35 agents, release agents, coating agents, Sweetening, flavoring Thus, in one aspect, the invention provides methods for and perfuming agents, preservatives, and antioxidants can reducing or inhibiting spermatozoa emission involving also be present in the compositions. administering an effective amount of a BRDT inhibitor to a Examples of pharmaceutically-acceptable antioxidants male Subject. In embodiments, the inhibitor is a compound include, but are not limited to: (1) water soluble antioxidants, having a formula delineated herein, a derivative thereof, or a 40 Such as ascorbic acid, cysteine hydrochloride, sodium bisul pharmaceutically acceptable salt or prodrug thereof. fate, sodium metabisulfite, sodium sulfite and the like; (2) In embodiments, the methods involve administering the oil-soluble antioxidants, such as ascorbyl palmitate, buty inhibitorin an amount Sufficient to Suppress spermatogenesis. lated hydroxyanisole (BHA), butylated hydroxytoluene In embodiments, the methods involve administering the (BHT), lecithin, propyl gallate, alpha-tocopherol, and the inhibitor in an amount Sufficient to induce azoospermia or 45 like; and (3) metal chelating agents, such as citric acid, eth oligoZoospermia. ylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, In embodiments, the methods involve administering the phosphoric acid, and the like. inhibitor in an amount Sufficient to lower the spermatozoa In embodiments, the pharmaceutical composition is pro concentration to not more than 3 million/mL, 2 million/mL, 1 vided in a solid form, such as a lyophilized powder suitable million/mL, 0.5 million/mL, 0.25 million/mL, or 0.1 million/ 50 for reconstitution, a liquid Solution, Suspension, emulsion, mL. In related embodiments, the methods involve adminis tablet, pill, capsule, Sustained release formulation, or powder. tering the inhibitor in an amount sufficient to lower the sper In embodiments, the pharmaceutical composition is Sup matozoa concentration to not more than 0.1 million/mL. plied in liquid form, for example, in a sealed container indi In embodiments, the inhibitor is adminstered in combina cating the quantity and concentration of the active ingredient tion with a pharmaceutically acceptable carrier, excipient, or 55 in the pharmaceutical composition. In related embodiments, diluent. the liquid form of the pharmaceutical composition is Supplied In embodiments, the inhibitor is administered to the subject in a hermetically sealed container. orally, transdermally, or by injection. In related embodi Methods for formulating the pharmaceutical compositions ments, the inhibitor is administered in the form of a tablet or of the present invention are conventional and well-known in capsule. In related embodiments, the inhibitor is administered 60 the art (see Remington and Remington's). One of skill in the by parenteral injection, intramuscular injection, intravenous art can readily formulate a pharmaceutical composition hav injection, Subcutaneous implantation, Subcutaneous injec ing the desired characteristics (e.g., route of administration, tion, or transdermal preparation. biosafety, and release profile). In embodiments, the inhibitor is used in combination with Methods for preparing the pharmaceutical compositions at least one additional male contraceptive agent or device. In 65 include the step of bringing into association the active ingre related embodiments, the additional male contraceptive is a dient with a pharmaceutically acceptable carrier and, option condom. In other related embodiments, the additional male ally, one or more accessory ingredients. The pharmaceutical US 9,301.962 B2 83 84 compositions can be prepared by uniformly and intimately made by molding in a suitable machine a mixture of the bringing into association the active ingredient with liquid powdered active ingredient moistened with an inert liquid carriers, or finely divided solid carriers, or both, and then, if diluent. necessary, shaping the product. Additional methodology for The tablets and other Solid dosage forms, such as dragees, preparing the pharmaceutical compositions, including the capsules, pills, and granules, can optionally be scored or preparation of multilayer dosage forms, are described in prepared with coatings and shells, such as enteric coatings Ansel's Pharmaceutical Dosage Forms and Drug Delivery and other coatings well-known in the art. Systems (9th ed., Lippincott Williams & Wilkins), which is The pharmaceutical compositions can also be formulated hereby incorporated by reference. so as to provide slow, extended, or controlled release of the 10 active ingredient therein using, for example, hydroxypropy Methods of Delivery lmethyl cellulose in varying proportions to provide the The pharmaceutical compositions of the present invention desired release profile, other polymer matrices, liposomes can be administered to a subject by oral and non-oral means and/or microspheres. The pharmaceutical compositions can (e.g., topically, transdermally, or by injection). Such modes of also optionally contain opacifying agents and may be of a administration and the methods for preparing an appropriate 15 composition that releases the active ingredient(s) only, or pharmaceutical composition for use therein are described in preferentially, in a certain portion of the gastrointestinal tract, Gibaldi's Drug Delivery Systems in Pharmaceutical Care optionally, in a delayed manner. Examples of embedding (1st ed., American Society of Health-System Pharmacists), compositions include polymeric Substances and waxes. The which is hereby incorporated by reference. active ingredient can also be in micro-encapsulated form, if In embodiments, the pharmaceutical compositions are appropriate, with one or more pharmaceutically acceptable administered orally in a solid form. carriers, excipients, or diluents well-known in the art (see, Pharmaceutical compositions Suitable for oral administra e.g., Remington and Remington's). tion can be in the form of capsules, cachets, pills, tablets, The pharmaceutical compositions can be sterilized by, for lozenges (using a flavored basis, usually Sucrose and acacia or example, filtration through a bacteria-retaining filter, or by tragacanth), powders, granules, or as a solution or a suspen 25 incorporating sterilizing agents in the form of sterile solid sion in an aqueous or non-aqueous liquid, or as an oil-in compositions which can be dissolved insterile water, or some water or water-in-oil liquid emulsion, or as an elixir or syrup, other sterile injectable medium immediately before use. or as pastilles (using an inert base. Such as gelatin and glyc In embodiments, the pharmaceutical compositions are erin, or Sucrose and acacia) and/or as mouth washes and the administered orally in a liquid form. 30 Liquid dosage forms for oral administration of an active like, each containing a predetermined amount of a ingredient include pharmaceutically acceptable emulsions, compound(s) described herein, a derivative thereof, or a phar microemulsions, Solutions, Suspensions, syrups and elixirs. maceutically acceptable salt or prodrug thereof as the active In addition to the active ingredient, the liquid dosage forms ingredient(s). The active ingredient can also be administered can contain inert diluents commonly used in the art, such as, as a bolus, electuary, or paste. 35 for example, water or other solvents, solubilizing agents and In solid dosage forms for oral administration (e.g., cap emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl Sules, tablets, pills, dragees, powders, granules and the like), carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, the active ingredient is mixed with one or more pharmaceu propylene glycol, 1,3-butylene glycol, oils (in particular, cot tically acceptable carriers, excipients, or diluents, such as tonseed, groundnut, corn, germ, olive, castor and sesame Sodium citrate or dicalcium phosphate, and/or any of the 40 oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols following: (1) fillers or extenders, such as starches, lactose, and fatty acid esters of sorbitan, and mixtures thereof. In Sucrose, glucose, mannitol, and/or silicic acid; (2) binders, addition to inert diluents, the liquid pharmaceutical compo Such as, for example, carboxymethylcellulose, alginates, sitions can include adjuvants such as wetting agents, emulsi gelatin, polyvinyl pyrrolidone, Sucrose and/or acacia; (3) fying and Suspending agents, Sweetening, flavoring, coloring, humectants, such as glycerol; (4) disintegrating agents, such 45 perfuming and preservative agents, and the like. as agar-agar, calcium carbonate, potato or tapioca starch, Suspensions, in addition to the active ingredient(s) can alginic acid, certain silicates, and sodium carbonate; (5) Solu contain Suspending agents such as, but not limited to, ethoxy tion retarding agents. Such as paraffin; (6) absorption accel lated isostearyl alcohols, polyoxyethylene sorbitol and sorbi erators, such as quaternary ammonium compounds; (7) wet tan esters, microcrystalline cellulose, aluminum metahydrox ting agents, such as, for example, acetyl alcohol and glycerol 50 ide, bentonite, agar-agar and tragacanth, and mixtures monostearate; (8) absorbents, such as kaolin and bentonite thereof. clay; (9) lubricants, such a talc, calcium Stearate, magnesium In embodiments, the pharmaceutical compositions are Stearate, Solid polyethylene glycols, Sodium lauryl Sulfate, administered by non-oral means such as by topical applica and mixtures thereof, and (10) coloring agents. In the case of tion, transdermal application, injection, and the like. In capsules, tablets, and pills, the pharmaceutical compositions 55 related embodiments, the pharmaceutical compositions are can also comprise buffering agents. Solid compositions of a administered parenterally by injection, infusion, or implan similar type can also be prepared using fillers in Soft and tation (e.g., intravenous, intramuscular, intraarticular, Subcu hard-filled gelatin capsules, and excipients such as lactose or taneous, and the like). milk Sugars, as well as high molecular weight polyethylene Compositions for parenteral use can be presented in unit glycols and the like. 60 dosage forms, e.g. in ampoules or in vials containing several A tablet can be made by compression or molding, option doses, and in which a suitable preservative can be added. Such ally with one or more accessory ingredients. Compressed compositions can be in form of a solution, a Suspension, an tablets can be prepared using binders (for example, gelatin or emulsion, an infusion device, a delivery device for implanta hydroxypropylmethyl cellulose), lubricants, inert diluents, tion, or it can be presented as a dry powder to be reconstituted preservatives, disintegrants (for example, sodium starch gly 65 with water or another suitable vehicle before use. One or more colate or cross-linked sodium carboxymethyl cellulose), Sur co-vehicles, such as ethanol, can also be employed. Apart face-actives, and/or dispersing agents. Molded tablets can be from the active ingredient(s), the compositions can contain US 9,301.962 B2 85 86 Suitable parenterally acceptable carriers and/or excipients or Materials for use in implants can be non-biodegradable, the active ingredient(s) can be incorporated into micro e.g., polydimethylsiloxane, or biodegradable Such as, e.g., spheres, microcapsules, nanoparticles, liposomes, or the like poly(caprolactone), poly(lactic acid), poly(glycolic acid) or for controlled release. Furthermore, the compositions can poly(ortho esters). also contain Suspending, solubilising, stabilising, pH-adjust 5 In embodiments, the active ingredient(s) are administered ing 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 can 10 also be administered using a Sonic nebulizer, which would able liquid vehicle. Exemplary vehicles and solvents include, minimize exposing the agent to shear, which can result in but are not limited to, water, water adjusted to a suitable pH by degradation of the compound. addition of an appropriate amount of hydrochloric acid, Ordinarily, an aqueous aerosol is made by formulating an sodium hydroxide or a suitable buffer, 1,3-butanediol, Ring aqueous solution or Suspension of the active ingredient(s) er's Solution and isotonic sodium chloride solution. The phar 15 together with conventional pharmaceutically-acceptable car maceutical composition can also contain one or more preser riers and stabilizers. The carriers and stabilizers vary with the Vatives, for example, methyl, ethyl or n-propyl requirements of the particular compound, but typically p-hydroxybenzoate. To improve solubility, a dissolution include nonionic Surfactants (Tweens, Pluronics, or polyeth enhancing or solubilising agent can be added or the solvent ylene glycol), innocuous proteins like serum albumin, Sorbi can contain 10-60% w/w of propylene glycol or the like. tan esters, oleic acid, lecithin, amino acids such as glycine, The pharmaceutical compositions can contain one or more buffers, salts, Sugars or Sugar alcohols. Aerosols generally are pharmaceutically acceptable sterile isotonic aqueous or non prepared from isotonic solutions. aqueous solutions, dispersions, Suspensions or emulsions, or Dosage forms for topical or transdermal administration of sterile powders, which can be reconstituted into sterile inject an active ingredient(s) includes powders, sprays, ointments, able solutions or dispersions just prior to use. Such pharma 25 pastes, creams, lotions, gels, solutions, patches and inhalants. ceutical compositions can contain antioxidants; buffers; bac The active ingredient(s) can be mixed understerile conditions teriostats; solutes, which render the formulation isotonic with with a pharmaceutically acceptable carrier, and with any pre the blood of the intended recipient; Suspending agents; thick servatives, buffers, or propellants as appropriate. ening agents; preservatives; and the like. Transdermal patches Suitable for use in the present inven Examples of Suitable aqueous and nonaqueous carriers, 30 tion are disclosed in Transdermal Drug Delivery. Develop which can be employed in the pharmaceutical compositions mental Issues and Research Initiatives (Marcel Dekker Inc., of the invention include water, ethanol, polyols (such as glyc 1989) and U.S. Pat. Nos. 4,743,249, 4,906,169, 5,198,223, erol, propylene glycol, polyethylene glycol, and the like), and 4,816,540, 5,422,119, 5,023,084, which are hereby incorpo Suitable mixtures thereof, vegetable oils, such as olive oil, and rated by reference. The transdermal patch can also be any injectable organic esters, such as ethyl oleate. Proper fluidity 35 transdermal patch well-known in the art, including transscro can be maintained, for example, by the use of coating mate tal patches. Pharmaceutical compositions in Such transdermal rials, such as lecithin, by the maintenance of the required patches can contain one or more absorption enhancers or skin particle size in the case of dispersions, and by the use of permeation enhancers well-known in the art (see, e.g., U.S. Surfactants. Pat. Nos. 4,379,454 and 4,973,468, which are hereby incor In some embodiments, in order to prolong the effect of an 40 porated by reference). Transdermal therapeutic systems for active ingredient, it is desirable to slow the absorption of the use in the present invention can be based on iontophoresis, compound from Subcutaneous or intramuscular injection. diffusion, or a combination of these two effects. This can be accomplished by the use of a liquid Suspension of Transdermal patches have the added advantage of provid crystalline or amorphous material having poor water solubil ing controlled delivery of active ingredient(s) to the body. ity. The rate of absorption of the active ingredient then 45 Such dosage forms can be made by dissolving or dispersing depends upon its rate of dissolution which, in turn, can the active ingredient(s) in a proper medium. Absorption depend upon crystal size and crystalline form. Alternatively, enhancers can also be used to increase the flux of the active delayed absorption of a parenterally-administered active ingredient across the skin. The rate of Such flux can be con ingredient is accomplished by dissolving or Suspending the trolled by either providing a rate controlling membrane or compound in an oil vehicle. In addition, prolonged absorption 50 dispersing the active ingredient(s) in a polymer matrix or gel. of the injectable pharmaceutical form can be brought about Such pharmaceutical compositions can be in the form of by the inclusion of agents that delay absorption Such as alu creams, ointments, lotions, liniments, gels, hydrogels, solu minum monostearate and gelatin. tions, Suspensions, sticks, sprays, pastes, plasters and other Controlled release parenteral compositions can be in form kinds of transdermal drug delivery systems. The composi of aqueous Suspensions, microspheres, microcapsules, mag 55 tions can also include pharmaceutically acceptable carriers or netic microspheres, oil solutions, oil Suspensions, emulsions, excipients such as emulsifying agents, antioxidants, buffer or the active ingredient can be incorporated in biocompatible ing agents, preservatives, humectants, penetration enhancers, carrier(s), liposomes, nanoparticles, implants or infusion chelating agents, gel-forming agents, ointment bases, per devices. fumes, and skin protective agents. Materials for use in the preparation of microspheres and/or 60 Examples of emulsifying agents include, but are not lim microcapsules include biodegradable/bioerodible polymers ited to, naturally occurring gums, e.g. gum acacia or gum Such as polyglactin, poly-(isobutyl cyanoacrylate), poly(2- tragacanth, naturally occurring phosphatides, e.g. soybean hydroxyethyl-L-glutamine) and poly(lactic acid). lecithin and sorbitan monooleate derivatives. Biocompatible carriers which can be used when formulat Examples of antioxidants include, but are not limited to, ing a controlled release parenteral formulation include car 65 butylated hydroxy anisole (BHA), ascorbic acid and deriva bohydrates Such as dextrans, proteins such as albumin, lipo tives thereof, tocopherol and derivatives thereof, and cys proteins or antibodies. teine. US 9,301.962 B2 87 88 Examples of preservatives include, but are not limited to, Subcutaneous implant devices can be slow-release cap parabens, such as methyl or propyl p-hydroxybenzoate and Sules made with any suitable polymer, e.g., as described in benzalkonium chloride. U.S. Pat. Nos. 5,035,891 and 4.210,644, which are hereby Examples of humectants include, but are not limited to, incorporated by reference. glycerin, propylene glycol, Sorbitol and urea. In general, at least four different approaches are applicable Examples of penetration enhancers include, but are not in order to provide rate control over the release and transder limited to, propylene glycol, DMSO, triethanolamine, N.N- mal permeation of a drug compound. These approaches are: dimethylacetamide, N,N-dimethylformamide, 2-pyrrolidone membrane-moderated systems, adhesive diffusion-con and derivatives thereof, tetrahydrofurfuryl alcohol, propylene trolled systems, matrix dispersion-type systems and 10 microreservoir systems. It is appreciated that a controlled glycol, diethylene glycol monoethyl or monomethyl ether release percutaneous and/or topical composition can be with propylene glycol monolaurate or methyl laurate, euca obtained by using a suitable mixture of these approaches. lyptol, lecithin, Transcutol R, and AZone(R). In a membrane-moderated system, the active ingredient is Examples of chelating agents include, but are not limited present in a reservoir which is totally encapsulated in a shal to, Sodium EDTA, citric acid and phosphoric acid. 15 low compartment molded from a drug-impermeable lami Examples of gel forming agents include, but are not limited nate, such as a metallic plastic laminate, and a rate-control to, Carbopol, cellulose derivatives, bentonite, alginates, gela ling polymeric membrane Such as a microporous or a non tin and polyvinylpyrrolidone. porous polymeric membrane, e.g., ethylene-vinyl acetate In addition to the active ingredient(s), the ointments, copolymer. The active ingredient is released through the rate pastes, creams, and gels of the present invention can contain controlling polymeric membrane. In the drug reservoir, the excipients, such as animal and vegetable fats, oils, waxes, active ingredient can either be dispersed in a solid polymer paraffins, starch, tragacanth, cellulose derivatives, polyethyl matrix or Suspended in an unleachable, Viscous liquid ene glycols, silicones, bentonites, silicic acid, talc and Zinc medium such as silicone fluid. On the external surface of the oxide, or mixtures thereof. polymeric membrane, a thin layer of an adhesive polymer is Powders and sprays can contain excipients such as lactose, 25 applied to achieve an intimate contact of the transdermal talc, silicic acid, aluminum hydroxide, calcium silicates and system with the skin surface. The adhesive polymer is pref polyamide powder, or mixtures of these Substances. Sprays erably a polymer which is hypoallergenic and compatible can additionally contain customary propellants. Such as chlo with the active drug Substance. rofluorohydrocarbons, and volatile unsubstituted hydrocar In an adhesive diffusion-controlled system, a reservoir of bons, such as butane and propane. 30 the active ingredient is formed by directly dispersing the Injectable depot forms are made by forming microencap active ingredient in an adhesive polymer and then by, e.g., sule matrices of compound(s) of the invention in biodegrad solvent casting, spreading the adhesive containing the active able polymers such as polylactide-polyglycolide. Depending ingredient ance onto a flat sheet of Substantially drug-imper on the ratio of compound to polymer, and the nature of the meable metallic plastic backing to form a thin drug reservoir particular polymer employed, the rate of compound release 35 layer. can be controlled. Examples of other biodegradable polymers A matrix dispersion-type system is characterized in that a include poly(orthoesters) and poly(anhydrides). Depot reservoir of the active ingredient is formed by substantially injectable formulations are also prepared by entrapping the homogeneously dispersing the active ingredient in a hydro drug in liposomes or microemulsions which are compatible philic or lipophilic polymer matrix. The drug-containing with body tissue. 40 polymer is then molded into disc with a substantially well Subcutaneous implants are well-known in the art and are defined surface area and controlled thickness. The adhesive suitable for use in the present invention. Subcutaneous polymer is spread along the circumference to form a strip of implantation methods are preferably non-irritating and adhesive around the disc. mechanically resilient. The implants can be of matrix type, of A microreservoir system can be considered as a combina reservoir type, or hybrids thereof. In matrix type devices, the 45 tion of the reservoir and matrix dispersion type systems. In carrier material can be porous or non-porous, Solid or semi this case, the reservoir of the active substance is formed by Solid, and permeable or impermeable to the active compound first Suspending the drug Solids in an aqueous Solution of or compounds. The carrier material can be biodegradable or water-soluble polymer and then dispersing the drug Suspen may slowly erode after administration. In some instances, the sion in a lipophilic polymer to form a multiplicity of unleach matrix is non-degradable but instead relies on the diffusion of 50 able, microscopic spheres of drug reservoirs. the active compound through the matrix for the carrier mate Any of the above-described controlled release, extended rial to degrade. Alternative subcutaneous implant methods release, and Sustained release compositions can be formu utilize reservoir devices where the active compound or com lated to release the active ingredient in about 30 minutes to pounds are Surrounded by a rate controlling membrane, e.g., about 1 week, in about 30 minutes to about 72 hours, in about a membrane independent of component concentration (pos 55 30 minutes to 24 hours, in about 30 minutes to 12 hours, in sessing Zero-order kinetics). Devices consisting of a matrix about 30 minutes to 6 hours, in about 30 minutes to 4 hours, Surrounded by a rate controlling membrane also suitable for and in about 3 hours to 10 hours. In embodiments, an effective SC. concentration of the active ingredient(s) is Sustained in a Both reservoir and matrix type devices can contain mate subject for 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 rials such as polydimethylsiloxane, such as SilasticTM, or 60 hours, 24 hours, 48 hours, 72 hours, or more after adminis other silicone rubbers. Matrix materials can be insoluble tration of the pharmaceutical compositions to the Subject. polypropylene, polyethylene, polyvinyl chloride, ethylvinyl Methods of Delivery acetate, polystyrene and polymethacrylate, as well as glyc When the compound(s) of the invention are administered erol esters of the glycerol palmitostearate, glycerol Stearate, as pharmaceuticals to humans and animals, they can be given and glycerol behenate type. Materials can be hydrophobic or 65 perse or as a pharmaceutical composition containing active hydrophilic polymers and optionally contain solubilising ingredient in combination with a pharmaceutically accept agents. able carrier, excipient, or diluent. US 9,301.962 B2 89 90 Actual dosage levels and time course of administration of macy, 20th and 21st Editions, Gennaro and University of the the active ingredients in the pharmaceutical compositions of Sciences in Philadelphia, Eds. Lippencott Williams & the invention can be varied so as to obtain an amount of the Wilkins (2003 and 2005), which are hereby incorporated by active ingredient which is effective to achieve the desired reference. therapeutic response for a particular patient, composition, Kits and mode of administration, without being toxic to the The invention provides for a kit for effecting male contra patient. Generally, compounds or pharmaceutical composi ception. In embodiments, the kit contains one or more of the tions of the invention are administered in an effective amount compounds or pharmaceutical compositions described or quantity Sufficient to reduce or inhibit spermatozoa emis herein. In embodiments, the kit provides instructions for use. sion in a male Subject. In embodiments, administration of the 10 compound or pharmaceutical composition Suppresses sper The instructions for use can pertain to any of the methods described herein. In related embodiments, the instructions matogenesis, induces azoospermia, or induces oligoZoosper pertain to using the compound(s) or pharmaceutical compo 18. Exemplary dose ranges include 0.01 mg to 250 mg per day, sition(s) for reducing or inhibiting spermatozoa emission. In 0.01 mg to 100 mg per day, 1 mg to 100 mg per day, 10 mg to 15 embodiments, the kit provides a notice in the form prescribed 100 mg per day, 1 mg to 10 mg per day, and 0.01 mg to 10 mg by a governmental agency regulating the manufacture, use, or per day. A preferred dose of the compound of the invention is sale of pharmaceuticals or biological products, which notice the maximum that a patient can tolerate and not develop reflects approval by the agency of manufacture, use or sale of serious or unacceptable side effects. In embodiments, the the kit and the components therein for human administration. compound(s) of the present invention is administered at a The invention also provides for compound(s) or pharma concentration of about 10 micrograms to about 100 mg per ceutical composition(s) packaged in a hermetically sealed kilogram of body weight per day, about 0.1 to about 10 mg/kg container (e.g., ampoule or Sachette) indicating the quantity per day, or about 1.0 mg to about 10 mg/kg of body weight per of compound. In embodiments, a compound or pharmaceu day. In embodiments, the pharmaceutical composition com tical composition is Supplied as a liquid. In other embodi prises a compound(s) of the invention in an amount ranging 25 ments, a compound or pharmaceutical composition is Sup between 1 and 10 mg, such as 1,2,3,4,5,6,7,8,9, or 10 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 composi concentration for administration to a Subject. tions typically should provide a dosage of from about 0.001 30 The invention also provides for transdermal patches con mg to about 2000 mg of compound per kilogram of body taining the compound(s) or pharmaceutical composition(s). weight per day. For example, dosages for systemic adminis In embodiments, the kit provides compound(s) or pharma tration to a human patient can range from 1-10 ug/kg, 20-80 ceutical composition(s) in more than one dosage unit. The kit ug/kg, 5-50 g/kg, 75-150 g/kg, 100-500 ug/kg, 250-750 can contain from 1 to about 120 or more, from 1 to about 60, ug/kg, 500-1000 g/kg, 1-10 mg/kg, 5-50 mg/kg, 25-75 35 from 1 to about 30, from 1 to about 10, or from 1 to about 7 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 50-100 mg/kg, 250 dosage units. In cases where the compound(s) or pharmaceu 500 mg/kg, 500-750 mg/kg, 750-1000 mg/kg, 1000-1500 tical composition(s) is adapted to release a therapeutically mg/kg, 1500-2000 mg/kg, 5 mg/kg, 20 mg/kg, 50 mg/kg, 100 effective amount of the active ingredient over a 24 hour mg/kg, 500 mg/kg, 1000 mg/kg, 1500 mg/kg, or 2000 mg/kg. period, the kit conveniently comprises 1, about 5, about 7. Pharmaceutical dosage unit forms are prepared to provide 40 about 10, about 14, or about 30 dosage units. In cases where from about 1 mg to about 5000 mg, for example from about the compound(s) or pharmaceutical composition(s) is 100 to about 2500 mg of the compound or a combination of adapted to provide a therapeutically effective amount of the essential ingredients per dosage unit form. active ingredient over a 12 hour period, the kit conveniently In embodiments, the pharmaceutical composition com comprises 1, 2, about 10, about 14, about 30 or about 60 prises a compound(s) of the invention in an amount Sufficient 45 dosage units. In cases where the compound(s) or pharmaceu to lower spermatozoa concentration to not more than 3 mil tical composition(s) is adapted to provide a therapeutically lion/mL of semen, such as not more than 2 million/mL, 1 effective amount of the active ingredient over an about 3 to million/mL, 0.5 million/mL, 0.25 million/mL, or 0.1 million/ about 10 hour (e.g., about a 6 or 8 hour) period, the kit mL. In related embodiments, the pharmaceutical composi comprises about 1, about 4, about 40, about 60 or about 120 tion comprises a compound(s) of the invention in an amount 50 dosage units. One skilled in the art will recognize that other Sufficient to lower spermatozoa concentration to not more numbers of dosage units can be included in the kit without 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 As described herein, the invention provides specific detailed disclosure provided herein. Generally, an efficacious 55 examples of chemical compounds, including JQ1, as well as or effective amount of a compound(s) of the invention is other substituted compounds that bind a bromodomain bind determined by first administering a low dose of the ing pocket and are useful as a male contraceptive. However, compound(s) and then incrementally increasing the adminis the invention is not so limited. The invention further provides tered dose or dosages until a desired effect (e.g., decreased a simple means for identifying agents (including nucleic spermatozoa levels in seminal fluid) is observed in the treated 60 acids, peptides, Small molecule inhibitors, and mimetics) that Subject, with minimal or acceptable toxic side effects. Appli are capable of inhibiting spermatogenesis. Such compounds cable methods for determining an appropriate dose and dos are also expected to be useful as male contraceptives. ing schedule for administration of a pharmaceutical compo In particular embodiments, the effect of a compound or sition of the present invention are described, for example, in other agent of the invention is analyzed by assaying sper Goodman and Gilman's The Pharmacological Basis of 65 matogenesis. Agents and compounds of the invention that Therapeutics, Goodman et al., eds., 11th Edition, McGraw reduce spermatogenesis are identified as useful as male con Hill 2005, and Remington. The Science and Practice of Phar traceptives. US 9,301.962 B2 91 92 Virtually any agent that specifically binds to a BET family Alternatively, chemical compounds to be used as candidate member or that reduces the biological activity of a BET compounds can be synthesized from readily available starting family member may be employed in the methods of the inven materials using standard synthetic techniques and method tion. Methods of the invention are useful for the high ologies known to those of ordinary skill in the art. Synthetic throughput low-cost screening of candidate agents that 5 chemistry transformations and protecting group methodolo reduce or otherwise inhibit spermatogenesis. A candidate gies (protection and deprotection) useful in synthesizing the agent that specifically binds to a bromodomain of a BET compounds identified by the methods described herein are family member is then isolated and tested for activity in an in known in the art and include, for example, those such as vitro assay or in vivo assay for its ability to inhibit spermato described in R. Larock, Comprehensive Organic Transforma genesis. One skilled in the art appreciates that the effects of a 10 candidate agent on a cell is typically compared to a corre tions, VCH Publishers (1989); T. sponding control cell not contacted with the candidate agent. W. Greene and P. G. M. Wuts, Protective Groups in Thus, the screening methods include comparing spermatoge Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. nesis in a testes contacted by a candidate agent to the sper Fieser and M. Fieser, Fieser and Fieser’s Reagents for matogenesis present in an untreated control testes. 15 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 Synthe specifically bind to and/or antagonize a bromodomain) may sis, John Wiley and Sons (1995), and subsequent editions be used as male contraceptives. Potential bromodomain thereof. antagonists include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acid ligands, aptamers, and Libraries of compounds may be presented in Solution (e.g., antibodies that bind to a BET family member bromodomain Houghten, Biotechniques 13:412-421, 1992), or on beads and reduce its activity. Candidate agents may be tested for (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364: their ability to reduce spermatogenesis. 555-556, 1993), bacteria (Ladner, U.S. Pat. No. 5,223.409), Test Compounds and Extracts spores (Ladner U.S. Pat. No. 5,223.409), plasmids (Cullet al., In certain embodiments, BET family member antagonists 25 Proc Natl AcadSci USA 89:1865-1869, 1992) or on phage (e.g., agents that specifically bind and reduce the activity of a (Scott and Smith, Science 249:386–390, 1990; Devlin, Sci bromodomain) are identified from large libraries of natural ence 249:404–406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. product or synthetic (or semi-synthetic) extracts or chemical 87:6378-6382, 1990; Felici, J. Mol. Biol. 222:301-310, 1991; libraries or from polypeptide or nucleic acid libraries, accord Ladner Supra.). ing to methods known in the art. Those skilled in the field of 30 In addition, those skilled in the art of drug discovery and drug discovery and development will understand that the development readily understand that methods for dereplica precise source of test extracts or compounds is not critical to tion (e.g., taxonomic dereplication, biological dereplication, the screening procedure(s) of the invention. Virtually any and chemical dereplication, or any combination thereof) or number of unknown chemical extracts or compounds can be the elimination of replicates or repeats of materials already screened using the methods described herein. Examples of 35 Such extracts or compounds include, but are not limited to, known for their activity should be employed whenever pos plant-, fungal-, prokaryotic- or animal-based extracts, fer sible. mentation broths, and synthetic compounds, as well as the When a crude extract is found to have BET family member modification of existing polypeptides. bromodomain binding activity further fractionation of the Libraries of natural polypeptides in the form of bacterial, 40 positive lead extract is necessary to isolate molecular con fungal, plant, and animal extracts are commercially available stituents responsible for the observed effect. Thus, the goal of from a number of Sources, including Biotics (Sussex, UK), the extraction, fractionation, and purification process is the Xenova (Slough, UK), Harbor Branch Oceangraphics Insti careful characterization and identification of a chemical tute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, entity within the crude extract that reduces spermatogenesis. Mass.). Such polypeptides can be modified to include a pro 45 Methods of fractionation and purification of such heterog tein transduction domain using methods known in the art and enous extracts are known in the art. If desired, compounds described herein. In addition, natural and synthetically pro shown to be useful as therapeutics are chemically modified duced libraries are produced, if desired, according to methods according to methods known in the art. known in the art, e.g., by standard extraction and fractionation methods. Examples of methods for the synthesis of molecular 50 EXAMPLES libraries can be found in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909, 1993: Erb et al., Proc. Natl. Acad. Sci. USA 91:11422, 1994, Zuckermann et al., J. It should be appreciated that the invention should not be Med. Chem. 37:2678, 1994; Cho et al., Science 261:1303, construed to be limited to the examples that are now 1993: Carrell et al., Angew. Chem. Int. Ed. Engl. 33:2059, 55 described; rather, the invention should be construed to 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061, include any and all applications provided herein and all 1994; and Gallop et al., J. Med. Chem. 37: 1233, 1994. Fur equivalent variations within the skill of the ordinary artisan. thermore, if desired, any library or compound is readily modi fied using standard chemical, physical, or biochemical meth I. Chemical Examples ods. 60 Numerous methods are also available for generating ran dom or directed synthesis (e.g., semi-synthesis or total Syn Synthesis and Methods of Preparation thesis) of any number of polypeptides, chemical compounds, including, but not limited to, Saccharide-, lipid-, peptide-, and Compounds of the invention can be synthesized by meth nucleic acid-based compounds. Synthetic compound librar 65 ods described herein, and/or according to methods known to ies are commercially available from Brandon Associates one of ordinary skill in the art in view of the description (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). herein. US 9,301.962 B2 93 94 -continued Scheme S1. Synthesis of the racemic N-N O bromodomain inhibitor (+)-JQ1. Me l N y - CO Me O O NC S., morpholine -- -e- EtOH, 70° C. 70% 10
(2-amino-4,5-dimethylthiophen-3-yl)(4-chlorophenyl)methanone (S2) HCTU, i-PrNEt Her 15 DMF, 23° C. 90% The compound JQ1 was prepared according to the scheme shown above. Sulfur (220 mg, 6.9 mmol. 1.00 equiv) was added as a solid to a solution of 4-chlorobenzoyl acetonitrile S1 (1.24 g. 6.9 O 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 ethanol HN y YacoobNHFmoc (20 ml, 0.35 M) at 23°C.'. The mixture was then heated to
Piperidine 70° C. After 12 hours, the reaction mixture was cooled to 23° DMF, 23° C. 25 C. and poured into brine (100 ml). The aqueous layer was He 90% extracted with ethyl acetate (3x50ml). The combined organic layers were washed with brine (50 ml), were dried over anhy drous sodium Sulphate, were filtered, and were concentrated under reduced pressure. The residue was purified by flash 30 column chromatography (Combiflash RF system, 40 gram silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford O S2 (1.28 g., 70%) as a yellow solid. y-coo-Bu (S)-tert-Butyl-3-((9H-fluoren-9-yl)methoxy
HN NH2 35 carbonyl)amino)-4-3-(4-chlorobenzoyl)-4,5-dim AcOH, EtOH ethylthiophen-2-yl)amino-4-oxobutanoate (S3) 80° C. -e- 95% (2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethy laminium hexafluorophosphate (HCTU) (827 mg, 2.0 mmol. 40 2.00 equiv), and N,N-diisopropylethylamine (0.72 ml. 4.0 mmol. 4.00 equiv) were added sequentially to a solution of 9-fluorenylmethoxycarbonyl-aspartic acid B-tert-butyl ester Fmoc-Asp(Ot-Bu)-OH (864 mg, 2.1 mmol. 2.10 equiv) in N,N-dimethylformamide (1.5 ml, 1.0 M). The mixture was 45 then stirred at 23° C. for 5 min. S2 (266 mg, 1.0 mmol. 1 HN O equiv) was then added as a Solid. The reaction mixture was N Me PSs, NaHCO stirred at 23°C. After 16 hours, ethyl acetate (20 ml) and brine diglyme (20 ml) were added. The two layers were separated, and the S He 85°C. aqueous layer was extracted with ethyl acetate (2x20ml). The 65.9% 50 combined organic layers were washed with brine (30 ml), were dried over with anhydrous sodium sulphate, were fil tered, and were concentrated under reduced pressure. The residue was purified by flash column chromatography (Com biflash RF, 40 gram silica gel, gradient 0 to 100% ethyl 55 acetate-hexanes) to afford S3 (625 mg, 90%) as brown oil. (S)-tert-butyl 3-amino-4-((3-(4-chlorobenzoyl)-4,5- dimethylthiophen-2-yl)amino)-4-oxobutanoate (S4)
S Me O-> 23° C. 60 Compound S3 (560 mg. 0.85 mmol. 1 equiv) was dissolved 2) CH3C(OCH3), into 20% piperidine in DMF solution (4.0 ml, 0.22 M) at 23° Toluene, 120° C. C. After 30 min, ethyl acetate (20 ml) and brine (20 ml) were 85% (2-steps) added to the reaction mixture. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2x20 Cl 65 ml). The combined organic layers were washed with brine S6 (3x25ml), were dried over anhydrous sodium sulphate, were filtered, and were concentrated under reduced pressure. The US 9,301.962 B2 95 96 residue was purified by flash column chromatography (Com biflash RF system, 24 gram silica gel, gradient 0 to 100% Scheme S2. Synthesis of enantiomerically enriched (+)-JQ1. ethyl acetate-hexanes) to afford free amine S4 (370 mg.90%) as yellow solid. The enantiomeric purity dropped to 75% O (determined with Berger Supercritical Fluid Chromatogra Fmoc-Asp(Ot-Bu)-OH phy (SFC) using AS-H column). PyBOP, i-PrNEt His DMF, 23° C. (S)-tert-Butyl 2-(5-(4-chlorophenyl)-6,7-dimethyl-2- 7296 oxo-2,3-dihydro-1H-thieno 2,3-e 1,4-diazepin-3-yl) acetate (S5) Cl S2 O Amino ketone (S4) (280 mg, 0.63 mmol) was dissolved in 10% acetic acid ethanol solution (21 ml, 0.03 M). The reac y cool B HN NHFmoc 15 tion mixture was heated to 85°C. After 30 minutes, all sol Piperidine vents were removed under reduced pressure. The residue was DMF, 23° C. purified by flash column chromatography (Combiflash RF -e- system, 12 gram silica gel, gradient 0 to 100% ethyl acetate 90% hexanes) to afford compound S5 (241 mg, 95%) as white solid. Enantiomeric purity of S5 was 67% (determined with Berger Supercritical Fluid Chromatography (SFC) using an AS-H column).
tert-Butyl 2-(5-(4-chlorophenyl)-6,7-dimethyl-2- 25 y-coo-Bu thioxo-2,3-dihydro-1H-thieno2,3-e 1,4-diazepin-3- NH2 SiO2, Toluene yl)acetate (S6) 90° C. -e- 95% Phosphorus pentasulfide (222 mg, 1.0 mmol. 2.00 equiv), 30 sodium bicarbonate (168 mg, 2.0 mmol. 4.00 equiv) were added sequentially to a solution of S5 (210 mg, 0.5 mmol. 1 equiv) in diglyme (1.25 ml, 0.4M). The reaction mixture was heated to 90° C. After 16h, brine (20 ml) and ethylacetate (35 35 ml) were added. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (3x30ml). The combined organic layers were washed with brine (2x15 ml), were dried over anhydrous sodium sulphate, were filtered, and were concentrated under reduced pressure. The residue 40 He was purified by flash column chromatography (Combiflash CH3CONHNH2, n-BuOH, RF system, 24 gram silica gel, gradient 0 to 100% ethyl 90° C. acetate-hexanes) to afford S6 (141 mg, 65%) as brown solid 92% with recovered S5 (73 mg, 34%). 45 tert-Butyl 2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H thieno 3.2-f 1.2.4 triazolo 4.3-a 1,4-diazepin-6-yl) acetate (+).JQ1 Hydrazine (0.015 ml, 0.45 mmol. 1.25 equiv) was added to 50 a solution of S6 (158 mg, 0.36 mmol. 1 equiv) in THF (2.6 ml, 0.14M) at 0°C. The reaction mixture was warmed to 23°C., and stirred at 23°C. for 1 h. All solvents were removed under Me reduced pressure. The resulting hydrazine was used directly 55 without purification. The hydrazine was then dissolved in a 2:3 mixture of trimethyl orthoacetate and toluene (6 ml, 0.06 (S)-tert-Butyl-3-(((9H-fluoren-9-yl)methoxycarbonyl)amino)-4-3-(4- M). The reaction mixture was heated to 120° C. After 2 h, all chlorobenzoyl)-4,5-dimethylthiophen-2-yl)amino)-4-oxobutanoate (S3) the solvents were removed under reduced pressure. The resi due was purified by flash column chromatography (Combi 60 (Benzotriazol-1-yloxyl)tripyrrolidinophosphonium (Py flash system, 4 g silica gel, gradient 0 to 100% ethyl acetate BOP) (494 mg. 0.95 mmol. 0.95 equiv), N,N-diisopropyl hexanes) to afford JQ1 (140 mg. 85% in 2 steps) as white ethylamine (0.50 ml, 2.8 mmol. 2.75 equiv) were added solid. The reaction conditions further epimerized the stereo sequentially to a solution of 9-fluorenylmethoxycarbonyl genic center, resulting in the racemate, JO1 (determined with 65 aspartic acid 13-tert-butyl ester Fmoc-Asp(Ot-Bu)-OH (411 Berger Supercritical Fluid Chromatography (SFC) with an mg, 1.00 mmol. 1.0 equiv) in N,N-dimethylformamide (1.0 AS-H column). ml, 1.0 M). The mixture was then stirred at 23°C. for 5 min. US 9,301.962 B2 97 98 S2 (266 mg, 1.0 mmol. 1 equiv) was then added as solid. The (Agilent High Pressure Liquid Chromatography using an reaction mixture was stirred at 23°C. After 4 h, ethyl acetate OD-H column) to provide the S-enantiomer in greater than (20 ml) and brine (20 ml) were added. The two layers were 99% ee. separated, and the aqueous layer was extracted with ethyl H NMR (600 MHz, CDC1, 25°C.) & 7.39 (d. J=8.4 Hz, acetate (2x20 ml). The combined organic layers were washed 5 2H), 7.31 (d. J=8.4 Hz, 2H), 4.54 (t, J=6.6 MHz, 1H), 3.54 with brine, were dried over with anhydrous sodium sulphate, 3.52 (m, 2H), 2.66 (s.3H), 2.39 (s.3H), 1.67 (s.3H), 1.48 (s, 9H). were filtered, and were concentrated under reduced pressure. 'CNMR (150MHz, CDC1,25°C.) & 1710, 163.8, 155.7, The residue was purified by flash column chromatography 150.0, 136.9, 131.1, 130.9, 130.6, 130.3, 128.9, 81.2, 54.1, (Combiflash RF system, 40 gram silica gel, gradient 0 to 38.1, 28.4, 14.6, 13.5, 12.1. 100% ethyl acetate-hexanes) to afford S3 (452 mg, 72%) as 10 HRMS (ESI) calc’d for CHCINOS M+H": brown oil. 457.1460, found 457.1451 m/z. TLC (EtOAc), Rf 0.32 (UV) (S)-tert-butyl 3-amino-4-((3-(4-chlorobenzoyl)-4,5- C’, (c (0.5, CHCl) dimethylthiophen-2-yl)amino)-4-oxobutanoate (S4) (-)-JQ1 was synthesized in a similar manner, employing 15 Fmoc-D-Asp(Ot-Bu)-OH as a starting material, and was fur Compound S3 (310 mg, 0.47 mmol. 1 equiv) was dissolved ther purified by chiral preparative HPLC (Agilent High Pres into 20% piperidine in DMF solution (2.2 ml, 0.22 M) at 23° Sure Liquid Chromatography using an OD-H column) to C. After 30 min, ethyl acetate (20 ml) and brine (20 ml) were afford the R-enantiomer in greater than 99% ee. C°,-72 added to the reaction mixture. The two layers were separated, (c 0.5, CHC1) and the aqueous layer was extracted with ethyl acetate (2x20 Synthesis of Additional Compounds ml). The combined organic layers were washed with brine Additional compounds of the invention were prepared as (3x25ml), were dried over anhydrous sodium sulphate, were illustrated in Scheme S3. filtered, and were concentrated under reduced pressure. The residue was purified by flash column chromatography (Com biflash RF system, 24 gram silica gel, gradient 0 to 100% Scheme S3. Synthesis of hydrazine derivatives. ethyl acetate-hexane) to afford free amine S4 (184 mg.90%) 25 as yellow solid. The enantiomeric purity was 91% (checked with Berger Supercritical Fluid Chromatography (SFC) using an AS-H column). (S)-tert-Butyl 2-(5-(4-chlorophenyl)-6,7-dimethyl-2- 30 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 toluene (10 ml, 0.04M). Silica gel (300 mg) was added, and 35 the reaction mixture was heated to 90° C. After 3 h, the reaction mixture was cooled to 23° C. The silica gel was filtered, and washed with ethyl acetate. The combined filtrates S r were concentrated. The residue was purified by flash column (1), (+)-JQ1 chromatography (Combiflash RF system, 12 gram silica gel. 40 C gradient 0 to 100% ethyl acetate-hexanes) to afford com pound S5 (168 mg.95%) as white solid. Enantiomeric purity of S5 was 90% (determined with Berger Supercritical Fluid Chromatography (SFC) using an AS-H column). (S)-tert-Butyl 2-(4-(4-chlorophenyl)-2,3,9-trimethyl 45 et N. 6H-thieno3.2-f 1.2.4 triazolo 4.3-a 1,4-diazepin 6-yl)acetate (+).JQ1 / \ re Potassium tert-butoxide (1.0 M solution in THF, 0.3 ml, S N N O 50 0.30 mmol. 1.10 equiv) was added to a solution of S5 (114 -/N 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 at (2) 23°C. for 30 min. The reaction mixture was cooled to -78°C. Diethyl chlorophosphate (0.047 ml, 0.32 mmol. 1.20 equiv) C was added to reaction mixture’. The resulting mixture was 55 warmed to -10° C. over 45 min. Acetic hydrazide (30 mg. 0.40 mmol. 1.50 equiv) was added to reaction mixture. The reaction mixture was stirred at 23° C. After 1 h, 1-butanol (2.25 ml) was added to reaction mixture, which was heated to et N. 90° C. After 1 h, all solvents were removed under reduce pressure. The residue was purified with flash column chro 60 matography (Combiflash system, 4 g silica gel, gradient 0 to N 100% ethyl acetate-hexanes) to afford (+)-JQ1 (114 mg. ) N1 N 6 NH2 92%) as white solid with 90% enantiomeric purity (deter mined with Berger Supercritical Fluid Chromatography yN (SFC) using AS-H column, 85% hexanes-methanol, 210 nm, 65 to (R-enantiomer)=1.59 min, t (S-enantiomer)=3.67 min). (3) The product was further purified by chiral preparative HPLC US 9,301.962 B2 99 100 C -continued 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-hy droxybenzaldehyde yielded the hydrazone (4). 2N. Both hydrazide (3) and hydrazone (4) showed activity in at least one biological assay. / \ - N2 10 A library of compounds was prepared by reaction of the S N1 NN O hydrazide (3) with a variety of carbonyl-containing com y- pounds (see Table A, above). OH Additional compounds were prepared for use, e.g., as (4)4 15 probes for assay development. An exemplary synthesis is shown in Scheme S4, below.
Scheme S4. Synthesis of derivatives useful as probes.
C C
MeOCOCl; HCOOH, 23° C. NH-NH2 N -e- N -e- 859% 859%
/ S \ N N Ory- ) S (, N N Y-lO
-/N -/N
C
N FITC, EtOH, 23° C. er -- H 85% N / \ N NH2 S N N O
\s N/
C OH eN C Sr-OS O S Ny- N O H N CO2H O O For FITC assay US 9,301.962 B2 101 102 -continued C
eN EDC, HOBt, 23° C. Her / \ O- 85% S N N O
-/N
C
N 1).5% TFA, CHC1.95% He H 2) Biotin, EDC, HOBt, 23° C. / \ Or N N-1 n-1'N-1O no-1 S-1'N-1N-1O NHTrt 90% --- Y-> --- Y - Y - N N 6 y W
N W \ N-1N-1'N-1O no-1N-1'N-1NO S N1N 6 O -/N
For Alpha assay
Additional compounds were prepared as shown in the table below:
Compound MS M+H" Name Structure mi?z (Observed)
(S)-JQ1 457.1
US 9,301.962 B2 105 106 -continued
Compound MS M+H" Name Structure mi?z (Observed)
JQ7 N 579.0 21 N Y. / N
C
JQ8 494.1
leN H O NHX N
C
JQ10 SO1.1 1. O 2N N N S Qu eNS. Xy O
C
JQ11 511.1
C
US 9,301.962 B2 109 110 -continued
Compound Name Structure JQ18 No 487.1
N 21 N N N-/ S N III leN O O
C Chemical Formula: C2H27CINOS Exact Mass: 486.14924 Molecular Weight: 487.01418
JQ19 471.1
C Chemical Formula: C2H27CINO2S Exact Mass: 470.15432 Molecular Weight: 471.01478
JQ20 370.1
C JQI-II-023 Chemical Formula: CoHgCINS Exact Mass: 370.101.90 Molecular Weight:370.89896 US 9,301.962 B2 111 112 -continued
Compound Name Structure
4431
N ... I III
C JQI-II-024 Chemical Formula: C22H2CINOS Exact Mass: 442.12302 Molecular Weight: 442.96.162
456.1
Chemical Formula: C2H2CINO2S Exact Mass: 455.1434 Molecular Weight:456.0001
456.1
N ... I III
C Chemical Formula: C2H2CINO2S Exact Mass: 455.1434 Molecular Weight:456.0001 US 9,301.962 B2 113 114 -continued
Compound MS M+H" Name Structure mi?z (Observed)
JQ25 SO6.1
N N YyN-/ S N "v O -eN HN O
C Chemical Formula: C26H2CINO2S Exact Mass: 505.1339 Molecular Weight:506.0191
JQB 389.2
Chemical Formula: C23H4NO2 Exact Mass: 388.1899 Molecular Weight: 388.4623
JQ30 456.2
N 2 N N N-/ S
leN NH
O
C Chemical Formula: C23H2CINOS Exact Mass: 455.1547 Molecular Weight:456.0034 US 9,301.962 B2 115 116 -continued
Compound Name Structure
JQ31 456.2
e N N
C Chemical Formula: C2H2CINOS Exact Mass: 455.1547 Molecular Weight:456.0034
468.1
Chemical Formula: C2H17CIFNOS Exact Mass: 467,0794 Molecular Weight: 467.8951
S12.2 N- ( O
Chemical Formula: C25H23CINO2S Exact Mass: S12.1761 Molecular Weight: 513.0548 US 9,301.962 B2 117 118 -continued
Compound MS M+H" Name Structure mi?z (Observed)
JQ34 505.1
N N YyN s --( \ /
C Chemical Formula: C26H25CINOS Exact Mass: SO4.1499 Molecular Weight: 505.0343
r). / \ \ - - \ /
C Chemical Formula: C27HCIN,OS Exact Mass: 539.2234 Molecular Weight: 540.1232
Chemical Formula: C27HCIN,OS Exact Mass: 539.2234 Molecular Weight: 540.1232 US 9,301.962 B2 119 120 -continued
Compound MS M+H" Name Structure mi?z (Observed)
JQ37 424.2
Chemical Formula: C22HNO2S Exact Mass: 423.1729 Molecular Weight: 423.5312
JQ38 SO8.2
eN NH
C Chemical Formula: C2H2CIN,OS Exact Mass: SO7.1608 Molecular Weight:508.0382
JQ39 505.1
S S.
C Chemical Formula: C26H25CINOS Exact Mass: SO4.1499 Molecular Weight: 505.0343 US 9,301.962 B2 121 122 -continued
Compound MS M+H" Name Structure mi?z (Observed)
JQ40 S12.2
N N-yN N-/ S
C Chemical Formula: C2HCIN,OS Exact Mass: 511.1921 Molecular Weight: 512.0700
Chemical Formula: C27HCIN,OS Exact Mass: 539.2234 Molecular Weight: 540.1232
JQ42 441.2
N Yy21 N s --4 Y-Nu/Y-d O
F Chemical Formula: C23H25FNO2S Exact Mass: 440.1682 Molecular Weight: 440.5336 US 9,301.962 B2 123 124 -continued
Compound MS M+H" Name Structure mi?z (Observed)
JQ43 494.1 eN s Y--4 N ().\ i?
N -e-N NH O
C Chemical Formula: C2H2CIN,OS Exact Mass: 493.1452 Molecular Weight: 494.0117
JQ44 S13.2
eN HN
C Chemical Formula: C25H23CINO2S Exact Mass: S12.1761 Molecular Weight: 513.0548
JQ45 494.1
eN HN
C Chemical Formula: C2H2CIN,OS Exact Mass: 493.1452 Molecular Weight: 494.0117 US 9,301.962 B2 125 126 -continued
Compound MS M+H" Name Structure mi?z (Observed)
JQ46 499.2
N O 2 N / S N \ / N
C Chemical Formula: C2HCINOS Exact Mass: 498.1969 Molecular Weight: 499.0712
JQ47 626.3 y)
Chemical Formula: CHCIN7O2S Exact Mass: 625.2966 Molecular Weight: 626.2555
JQ48 471.2
Cl
Exact Mass: 470.1543 Molecular Weight: 471.0148 US 9,301.962 B2 127 128 -continued Compound MS M+H" Name Structure mi?z (Observed) JQ49 C 429.1
eN / \ \ S N N N O
-/N
Exact Mass: 428.1074 Molecular Weight: 428.9350 JQ50 C S4O.2
etN W \ n-rr S N NN O N N SN
Exact Mass: 539.2234 Molecular Weight: 540.1232 JQ51 O 667.2 ( O O N \ N / N o O \
Cl
JQI-II-114 Exact Mass: 666.1816 Molecular Weight: 667.1764 JQ52 S13.2
SN ro
Exact Mass: 512.2125 Molecular Weight: 513.0978 US 9,301.962 B2 130 -continued Compound Name Structure JQ53 C 400.1
e-N
Exact Mass: 399.1284 Molecular Weight: 399.9402 Spectral data for each compound were consistent with the assigned structure,
II. Biological Activity and Methods of Treatment intraperitoneal injections of JO1 (50 mg/kg/day) or vehicle 25 control over a 3- or 6-week period. After 3 weeks of treatment, Example 1 mice were either sacrificed or mated to females while con tinuing to receive JO1. The JO1-treated males universally JQ1 is an Inhibitor of BRDT were observed to have grossly smaller testes compared to the control males (FIG. 5A). At each time point, males treated The feasibility of targeting human bromodomains with 30 with JO1 experienced a marked and significant reduction in acetyl-lysine competitive Small molecules was recently testes volumes (FIG. 5B). Males treated from 3-6 weeks of established (Filippakopoulos et al., Nature 468: 1067 (2010)). age showed a reduction to 75.4% of control, males treated The index study identified a potent thienodiazepine inhibitor from 6-9 weeks of age showed a reduction to 54.7% of con ((+)-JQ1; FIG. 2A, K-90 nM) of the BET family co-activa trols, and males treated for 6 weeks with JQ1 (6-12 weeks of tor protein BRD4 (Filippakopoulos et al.), which is impli 35 age) showed the most dramatic reduction to 40.6% of the cated in the pathogenesis of cancer. Protein sequence align controls (FIG. 5B). Consistent with the reduction in testes ment of human BRD4(1) to human BRDT(1) reveals 81% volumes, the tubules of JQ1 treated males were narrower with identity and 89% similarity, including all surface residues a decrease in the amount and number of tubules that had predicted to contact (+)-JQ1 (FIGS. 1 and 3). Based on these obvious and abundant spermatozoa in their lumen (FIGS. insights and preliminary evidence of binding to BRDT(1) 40 5C-5F). Whereas an abundance of seminiferous tubules from established by differential scanning fluorimetry (Filippako the control mice were observed to be full of spermatozoa poulos et al.), the biochemical and functional effects of (+)- (FIGS. 5C and 5E), the number of tubules with spermatozoa JQ1 on BRDT(1) were evaluated. and the amount of spermatozoa in these tubules were reduced To assess competitive binding to BRDT(1), a homoge in the JQ1-treated males (FIGS.5D and 5F). Consistent with neous, luminescence proximity assay (alpha-screen), capable 45 the reduction in testes weights (FIG. 5), the most dramatic of quantifying binding of a synthetic, biotinylated tetra-acety findings in the JO1-treated males (6 weeks treatment) were lated histone 4 peptide (H4Kac4, residues 1-20) to recombi seminiferous tubule degeneration where few tubules con nant epitope-tagged BRDT(1) was employed. Dose-ranging tained significant numbers of mature spermatozoa (FIGS. 5E studies of (+)-JQ1 demonstrated potent inhibition of H4Kac4 and 5G). Histological analysis of the epididymides of JQ1 binding, with a half-maximum inhibitory concentration 50 treated males also showed a similar finding in which fewer (ICs) value of 11 nM (FIG. 2B). In contrast, the (-)-JQ1 sperm were observed in the epididymal lumen compared to stereoisomer was inactive for BRDT(1), establishing a ste the abundance observed in the control (FIGS. 5G and 5H). reospecific, ligand-competitive binding event. These results are consistent with the findings from a repeat study in which C57B6 mice were treated with JQ1 (FIGS. Example 2 55 8A-8C) To further characterize these defects, spermatozoa number JQ1 Inhibits BRDT Activity During was determined after 3 weeks of treatment (3-6 weeks of age). Spermatogenesis It was found that epididymal sperm number were reduced to 27.8% of the control while after 6 weeks of treatment, the To determine the possible consequences of blocking 60 sperm in the cauda epididymis of the JO1-treated mice were BRDT function in vivo, the spermatogenic effects of JO1 10.9% of the control (FIG. 6A). Furthermore, whereas 85% administered to male mice were evaluated. Murine BRDT(1) of the sperm from the cauda epididymis of the control showed exhibits 90% amino acid sequence identity and 95% similar progressive motility, JQ1 treatment resulted in only 5% of the ity to human BRDT(1), including all surface residues influ spermatozoa with progressive motility. Thus, JO1 treatment encing molecular recognition (FIG. 4). Supporting the valid 65 quantitatively reduced sperm number and qualitatively ity of using JQ1 in murine model systems. Juvenile or adult reduced sperm motility. These findings phenocopy those C57BL6/J/129S5 hybrid male mice were administered daily observed in mice deficient in BRDT(1) (Shang et al., Devel US 9,301.962 B2 131 132 opment 134:3507 (2007)). Furthermore, the testosterone pro matids) (Kleene et al., Dev. Biol. 105:71 (1984)) are 2.1-fold ducing intertubular Leydig cells of the testes of JO1-treated to 4.0-fold lower in the testes of mice treated with JQ1 versus males appeared to be histologically normal (FIG. 6), and control. Unlike the Brdt knockout studies (Shang et al., there appeared to be no defects in androgen actions in these Development 134:3507 (2007)) in which the pachytene sper mice since the testosterone-responsive seminal vesicles of 5 matocyte-expressed gene. Histlhlt, is upregulated, JO1 treat JQ1-treated males were grossly normal. Lastly, since JO1 had ment leads to a 2.6-fold downregulation of this gene in line a significant effect on the seminiferous tubule compartment, with the suppression of Ccnal. Consistent with these mRNA it must be capable of effectively crossing the blood:testis findings and the histological analysis described above, JQ1 boundary to alter spermatogenesis. treatment reduced the number of spermatids positive for tran 10 sition protein 1 (TNP1) (FIGS. 7B and 7C), which is Example 3 expressed in the nuclei of step 10-15 spermatids (Zhao et al., Biol. Reprod. 71:1016 (2004)). JQ1 is a Reversible Inhibitor of BRDT Activity A pharmacologic approach to male contraception remains To further evaluate the consequences of JQ1 on male fer 15 a longstanding challenge in medicine. The results described tility and fertilization potential, control (n=2) and JQ1-treated herein provide pharmacologic validation of the amino-termi (n-3) males treated for 3 weeks were housed with 2 females nal bromodomain of BRDT as a target for male contracep each and subjected to treatments for an additional 3 weeks. tion, using a highly potent and selective chemical probe. JO1 Whereas the control males impregnated all 4 females, JO1 emerges as a lead compound for a new class of drugs that can had a contraceptive effect on the males (one failed to impreg cross the blood:testis boundary, inhibit bromodomain activity nate the two females, whereas only 1 of 2 females in each of during spermatogenesis, impair sperm generation and motill the other two cages became pregnant). When these same ity, reduce the number of oocytes fertilized, and produce a males were test bred to superovulated females (2 females per reversible contraceptive effect in mammals. As human and cage), after 5 weeks of treatment, all females demonstrated mouse BRDT proteins are highly conserved and have nearly copulation plugs indicating that JQ1 did not alter mating 25 identical bromodomain pockets based on our structural pre behavior, consistent with normal testosterone-responsive tis dictions, these discoveries can be completely translated to sues in these males. Oocytes from these females were col men, and provide a novel and efficacious strategy for a male lected from their oviductal ampulla and cultured for 2 days to contraceptive. determine their developmental potential post-mating (FIG. The results reported herein were obtained using the follow 6B). Whereas the majority of the oocytes from females mated 30 ing methods and materials. to controls developed into 2 cell (72.8%) and 4 cell (70.1%) (+)-JQ' embryos, few of the oocytes from the females mated to JQ1 The direct-acting, small-molecule bromodomain inhibitor males developed into 2 cell (10.1%) or 4 cell (6.6%) embryos, was prepared as previously described (Filippakopoulos et al., consistent with their lower sperm counts, decreased motility, Nature 468: 1067 (2010)). and fertility defects. Importantly, the effects of JO1 on male 35 Protein Cloning, Expression and Purification fertility were found to be reversible. Following cessation of The N-terminal domain of human BRDT was cloned, JQ1, 6 of 6 JQ1-treated adult male mice sired two litters of expressed in E-Coli and purified as previously described (Fil offspring (7.25+/-0.58 pups per litter) within the first ensuing ippakopoulos et al.). month. These results are consistent with the findings that the BRDT Proximity Assay sperm motility, testes weight, and sperm count in male mice 40 Assays were performed with minor modifications from the returned towards normal levels after cessation of JO1 treat manufacturer's protocol (PerkinElmer, USA). All reagents ment (FIGS. 9A-9C). were diluted in 50 mM HEPES, 150 mM NaCl, 0.1% w/v BSA, 0.01% w/v. Tween20, pH 7.5 and allowed to equilibrate Example 4 to room temperature prior to addition to plates. After addition 45 of Alpha beads to master solutions all Subsequent steps were Molecular Analysis of JQ1 Mediated BRDT performed in low light conditions. A 2x Solution of compo Inhibition nents with final concentrations of BRDT at 80 nM, Ni-coated Acceptor Bead at 25 g/ml, and 80 nM biotinylated H4-tetra To molecularly define the stages of spermatogenesis at acetyl was added in 10 uI to 384-well plates (AlphaPlate which JQ1 functions, quantitative RT-PCR was performed on 50 384, PerkinElmer, USA). Biotinylated peptide for BRDT was testes isolated from JO1-treated mice and controls (FIG. 7A). synthesized in-house on a CEM Liberty 9008005 microwave Genes expressed early in spermatogenesis such as Plzf, which peptide synthesizer: H4-tetra acetyl, Biotin-PEG2 is a marker for spermatogonial stem cells and early dividing SGRGKacGGKacGLGKacGGAKacRHRK COOH. spermatogonia (Buaas et al., Nat. Genet. 36:647 (2004); and Addition to wells was performed with either a multichannel Costoya et al., Nat. Genet. 36:653 (2004)), and Stra&, which 55 pipet (for optimization experiments) or a Biotek EL406 liquid is expressed mainly in differentiating spermatogonia and pre handler. After a 1 minute 1000 rpm spin-down, 100 ml of leptotene spermatocytes (Zhou et al., Biol. Reprod. 79:35 compounds from Stock plates were added by pin transfer (2008)), are 2.0-fold and 1.3-fold enriched, respectively, in using a Janus Workstation (PerkinElmer, USA). The strepta the testes of JO1-treated mice compared to control males. vidin-coated donor beads (25ug/ml final) were added as with However, genes expressed during meiosis or spermiogenesis 60 previous solution in a 2x, 10ul volume. Following this addi including Brdt (expressed in mid- to late-spermatocytes) tion, the plates were sealed with foil to block light exposure (Shang et al., Gene Expr: Patterns 4:513 (2004)), Ccnal (ex and to prevent evaporation. The plates were spun down again pressed in pachytene spermatocytes) (Sweeney et al., Devel at 1000 rpm for 1 minute. Next, the plates were incubated in opment 122:53 (1996)), Papolb (expressed in step 1-7 round the room with the plate reader (for temperature equilibration) spermatids) (Kashiwabara et al., Dev. Biol. 228: 106 (2000)), 65 for 1.5 hour prior to reading the assay. AlphaScreen measure Klf17 (expressed in step 4-7 spermatids) (Yan et al., Mech. ments were performed on an Envision 2104 (PerkinElmer, Dev. 118:233 (2002)), and Prm 1 (expressed in step 7-16 sper USA) utilizing the manufacturer's protocol. US 9,301.962 B2 133 134 Sequence Alignment as described (Roy et al., Faseb J. 21:1013 (2007)). In brief, Amino acid sequences for full-length bromodomain-con epididymides were dissected and placed in prewarmed M2 taining proteins were obtained from the US National Heart, medium, minced, and incubated at 37°C. in a CO incubator Lung and Blood Institute (Human BRDT accession number prior to counting. Q58F21; Human BRD4 accession number 060885; Mouse Fertilization and Embryo Developmental Potential BRDT accession number Q91Y44). Multiple sequencealign ments of full-length BRDT and BRD4 were generated using To evaluate the ability of spermatozoa of treated mice to MAFFT (v6.240) (Katoh et al., Nucleic Acids Res. 33:511 mate with females and fertilize oocytes, 21-day-old C57BL6/ (2005); Katoh et al., Nucleic Acids Res. 30:3059 (2002); and J/129S5 hybrid females were injected with 5 IU of pregnant Katoh and Toh, Brief Bioinform. 9:286 (2008)). The E-INS-i 10 mare serum gonadotropin (PMSG; Calbiochem, EMD, Gibb algorithm was selected as Suitable for sequences containing stown, N.J.) followed by 5 IU of human chorionic gonadot potentially large unalignable regions, and the BLOSUM62 ropin (hCG, Calbiochem, EMD, Gibbstown, N.J.) 48 hours scoring matrix was used as Suitable for highly evolutionarily later and mated to treated males. Oocytes were isolated from conserved sequences. Gap opening penalty and offset value ampullas of Oviducts of females with copulation plugs, were set to default parameters. 15 counted, and cultured in M16 medium (Sigma-Aldrich, St. Mouse Studies Louis, Mo.) for 24 hours (for counting of 2 cell embryos) and (+)-JQ1 was dissolved in DMSO at 50 mg/ml and then 48 hours (for counting of 4 cell embryos) as described (An diluted 1:10 in (2-Hydroxypropyl)-f-cyclodextrin (Sigma dreu-Vieyra et al., PLoS Biol. 8:e1000453 (2010); and Burns Aldrich, St. Louis, Mo.). The subsequent mixture was et al., Science 300:633 (2003)). injected intraperitoneal into male mice at 1% of the body weight of the mouse (final amount is 50 mg/kg/day). The Quantitative RT-PCR Analysis control was DMSO dissolved 1:10 in (2-Hydroxypropyl)-3- Total RNAs from mouse testes were isolated using TRIZol cyclodextrin and injected similarly. Juvenile or adult reagent (Invitrogen, Carlsbad, Calif.). Total RNA was then C57BL6/J/12955 hybrid mice for these studies were weighed reversely transcribed using Superscript III reverse tran daily before injections and fed ad libitum. These studies were 25 scriptase (Invitrogen, Carlsbad, Calif.). Quantitative PCR approved by the Administrative Committee on Laboratory was performed using SYBR green master mix and custom Animal Care at Baylor College of Medicine, and all experi ized primers (Table 1). TABLE 1. Primers for duantitative PCR
Gene ale Forward Rewerse
PZf TGGAGAAGCATTTGGGTATCTACTC AAGACGGCATGCTCAACACA (SEO ID NO. 5) (SEQ ID NO : 6)
Stra.8 GAGTGAGGCCCAGCATATGTC CCTCTGGATTTTCTGAGTTGCA (SEO ID NO: 7) (SEQ ID NO: 8)
Brot GCTTTGGGACTCCACAACTACTATG GATTGTCCATTTTCCCCTTGATC (SEO ID NO: 9) (SEQ ID NO: 10)
Cona1 TTTCCCCAATGCTGGTTGA AACCAAAATCCGTTGCTTCCT (SEQ ID NO: 11) (SEQ ID NO: 12)
Hist1h1t GCTGATTCCTGAGGCCCTTT CAGGGCAGCAAGGGACAT (SEQ ID NO: 13) (SEQ ID NO: 14)
Papolb CGCCAACAGAGAAACAACATTTAG CCAACCAGGATTCGGATCTTT (SEQ D NO: 15) (SEQ ID NO: 16)
Klf17 CCTCCCGTTTGTTCT CAACTTG GGTGCATAGCCTGTTCCTTATTG (SEO ID NO: 17) (SEQ ID NO: 18)
Prm1. TGCACAGAATAGCAAGTCCATCA TGTGGCGAGATGCTCTTGAA (SEQ ID NO: 19) (SEQ ID NO: 2O) ments were conducted in accordance with the NIH guide for 55 All quantitative PCR assays were conducted in duplicate for the Care and Use of Laboratory Animals. each sample. Gapdh was used as an internal control for the Histological Analysis quantification. Histological analysis of Bouin’s fixed testes and epid Other Embodiments idymides was performed as previously described (Kumar et From the foregoing description, it will be apparent that al., Nature Genetics 15:201 (1997)) using Periodic acid 60 variations and modifications may be made to the invention Schiff and hematoxylin. Rabbit anti-TNP2 (1:600) staining described herein to adopt it to various usages and conditions. and hematoxylin counter-staining was performed as Such embodiments are also within the scope of the following described (Zhao et al., Biol. Reprod. 71:1016 (2004)) using claims. Bouin’s fixed testes. The recitation of a listing of elements in any definition of a Epididymal Sperm Counts 65 variable herein includes definitions of that variable as any Counts were performed on spermatozoa isolated from the single element or combination (or Subcombination) of listed entire epididymis or from the caudal epididymis of adult mice elements. The recitation of an embodiment herein includes US 9,301.962 B2 135 136 that embodiment as any single embodiment or in combination individually indicated to be incorporated by reference. The with any other embodiments or portions thereof. subject matter described herein may be related to subject All patents and publications mentioned in this specification matter of U.S. provisional applications 61/334,991, 61/370, are herein incorporated by reference to the same extent as if 745, and 61/375,663, each of which is incorporated herein by each independent patent and publication was specifically and this reference.
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 2O
<21 Os SEQ ID NO 1 &211s LENGTH: 8O1 212s. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs SEQUENCE: 1 Met Leu Gln Asn Val Thr Pro His Asn Lys Lieu Pro Gly Glu Gly Asn 1. 5 1O 15 Ala Gly Lieu. Lieu. Gly Lieu. Gly Pro Glu Ala Ala Ala Pro Gly Lys Arg 2O 25 3 O Ile Arg Llys Pro Ser Lieu. Leu Tyr Glu Gly Phe Glu Ser Pro Thr Met 35 4 O 45
Ala Ser Wall Pro Ala Lieu. Glin Lieu. Thir Pro Ala Asn. Pro Pro Pro Pro SO 55 60 Glu Val Ser Asn. Pro Llys Llys Pro Gly Arg Val Thr Asn Glin Lieu. Glin 65 70 7s 8O Tyr Lieu. His Llys Val Val Met Lys Ala Lieu. Trp Llys His Glin Phe Ala 85 90 95 Trp Pro Phe Arg Glin Pro Val Asp Ala Wall Lys Lieu. Gly Lieu Pro Asp 1OO 105 110 Tyr His Lys Ile Ile Lys Gln Pro Met Asp Met Gly Thr Ile Lys Arg 115 12O 125 Arg Lieu. Glu Asn. Asn Tyr Tyr Trp Ala Ala Ser Glu. Cys Met Glin Asp 13 O 135 14 O Phe Asn Thr Met Phe Thr Asn Cys Tyr Ile Tyr Asn Llys Pro Thr Asp 145 15 O 155 16 O Asp Ile Val Lieu Met Ala Glin Thr Lieu. Glu Lys Ile Phe Lieu Gln Lys 1.65 17 O 17s
Wall Ala Ser Met Pro Glin Glu Glu Glin Glu Lieu Val Val Thir Ile Pro 18O 185 190 Lys Asn. Ser His Llys Lys Gly Ala Lys Lieu Ala Ala Lieu. Glin Gly Ser 195 2 OO 2O5
Wall. Thir Ser Ala His Glin Wall Pro Ala Wal Ser Ser Wal Ser His Thir 210 215 22 O Ala Lieu. Tyr Thr Pro Pro Pro Glu Ile Pro Thir Thr Val Lieu. Asn Ile 225 23 O 235 24 O Pro His Pro Ser Val Ile Ser Ser Pro Leu Lleu Lys Ser Lieu. His Ser 245 25 O 255 Ala Gly Pro Pro Leu Lieu Ala Val Thr Ala Ala Pro Pro Ala Glin Pro 26 O 265 27 O Lieu Ala Lys Llys Lys Gly Wall Lys Arg Lys Ala Asp Thir Thir Thr Pro 27s 28O 285 Thr Pro Thr Ala Ile Leu Ala Pro Gly Ser Pro Ala Ser Pro Pro Gly 290 295 3OO Ser Lieu. Glu Pro Lys Ala Ala Arg Lieu Pro Pro Met Arg Arg Glu Ser 305 31 O 315 32O
Gly Arg Pro Ile Llys Pro Pro Arg Lys Asp Lieu Pro Asp Ser Glin Glin 3.25 33 O 335 US 9,301.962 B2 137 138 - Continued
Glin His Glin Ser Ser Gly Lys Luell Ser Glu Glin Luell Lys His 34 O 345 35. O
Asn Gly Ile Lell Glu Luell Luell Ser Lys His Ala Ala Tyr 355 360 365
Ala Trp Pro Phe Tyr Pro Wall Asp Ala Ser Ala Lell Gly Luell His 37 O 375 38O
Asp His Asp Ile Ile His Pro Met Asp Lell Ser Thir Wall Lys 385 390 395 4 OO
Arg Met Glu Asn Arg Asp Arg Asp Ala Glin Glu Phe Ala Ala 4 OS 415
Asp Wall Arg Luell Met Phe Ser Asn Cys Asn Pro Pro Asp 425 43 O
His Asp Wall Wall Ala Met Ala Arg Luell Glin Asp Wall Phe Glu Phe 435 44 O 445
Arg Tyr Ala Met Pro Asp Glu Pro Luell Glu Pro Gly Pro Luell Pro 450 45.5 460
Wall Ser Thir Ala Met Pro Pro Gly Luell Ala Lys Ser Ser Ser Glu Ser 465 470
Ser Ser Glu Glu Ser Ser Ser Glu Ser Ser Ser Glu Glu Glu Glu Glu 485 490 495
Glu Asp Glu Glu Asp Glu Glu Glu Glu Glu Ser Glu Ser Ser Asp Ser SOO 505
Glu Glu Glu Arg Ala His Arg Luell Ala Glu Luell Glin Glu Glin Luell Arg 515 525
Ala Wall His Glu Glin Lell Ala Ala Luell Ser Glin Gly Pro Ile Ser Lys 53 O 535 54 O
Pro Arg Glu Glu Lys Arg Ala 5.45 550 555 560
Glu His Arg Gly Arg Ala Gly Ala Asp Glu Gly Pro 565 st O sts
Arg Ala Pro Arg Pro Pro Glin Pro Lys Ser Ala Ser Gly 585 59 O
Ser Gly Gly Gly Ser Ala Ala Luell Gly Pro Ser Gly Phe Gly Pro Ser 595 605
Gly Gly Ser Gly Thir Lell Pro Ala Thir Thir Ala Pro 610 615
Pro Ala Luell Pro Thir Gly Asp Ser Glu Glu Glu Glu Glu Ser Arg 625 630 635 64 O
Pro Met Ser Asp Glu Arg Glin Luell Ser Lell Asp Ile Asn Lys 645 650 655
Lell Pro Gly Glu Lys Lell Gly Arg Wall Wall His Ile Ile Glin Ala Arg 660 665 67 O
Glu Pro Ser Luell Arg Asp Ser Asn Pro Glu Glu Ile Glu Ile Asp Phe 675 685
Glu Thir Luell Pro Ser Thir Luell Arg Glu Luell Glu Arg Wall Luell 69 O. 695 7 OO
Ser Luell Arg Lys Lys Pro Arg Pro Tyr Thir Ile Pro 7 Os 71O 71s 72O
Wall Thir Lys Glu Glu Luell Ala Luell Glu Arg Glu Luell 72 73 O 73
Glu Arg Luell Glin Asp Wall Ser Gly Glin Luell Asn Ser Thir Lys 740 74. 7 O US 9,301.962 B2 139 140 - Continued
Pro Pro Lys Lys Ala Asn. Glu Lys Thir Glu Ser Ser Ser Ala Glin Glin 7ss 760 765
Wall Ala Wall Ser Arg Lieu. Ser Ala Ser Ser Ser Ser Ser Asp Ser Ser 770 775 78O
Ser Ser Ser Ser Ser Ser Ser Ser Ser Asp Thir Ser Asp Ser Asp Ser 78s 79 O 79. 8OO Gly
<210s, SEQ ID NO 2 &211s LENGTH: 726 212. TYPE : PRT &213s ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 2
Met Ser Thir Ala Thir Thir Wall Ala Pro Ala Gly Ile Pro Ala Thir Pro 1. 15
Gly Pro Wall Asn Pro Pro Pro Pro Glu Wall Ser Asn Pro Ser Pro 2O 25 3O
Gly Arg Lys Thir ASn Glin Lieu. Glin Met Glin Asn Wall Wall Wall 35 4 O 45
Thir Luell Trp His Glin Phe Ala Pro Phe Tyr Glin Pro Wall Asp SO 55 6 O
Ala Ile Luell Asn Lieu Pro Asp His Lys Ile Ile Asn Pro 65 70 8O
Met Asp Met Gly Thir Ile Llys Llys Arg Luell Glu Asn Asn Tyr Trp 85 90 95
Ser Ala Ser Glu Cys Met Glin Asp Phe Asn Thir Met Phe Thir Asn 105 11 O
Ile Tyr Asn Llys Pro Thr Asp Asp Ile Wall Lell Met Ala Glin Ala 115 12 O 125
Lell Glu Ile Phe Lieu Gln Lys Wall Ala Glin Met Pro Glin Glu Glu 13 O 135 14 O
Wall Glu Luell Luell Pro Pro Ala Pro Gly Lys Gly Arg Pro Ala 145 150 155 160
Ala Gly Ala Glin Ser Ala Gly Thr Glin Glin Wall Ala Ala Wall Ser Ser 1.65 17O 17s
Wall Ser Pro Ala Thr Pro Phe Glin Ser Wall Pro Pro Thir Wall Ser Glin 18O 185 19 O
Thir Pro Wall Ile Ala Ala Thr Pro Wall Pro Thir Ile Thir Ala Asn Wall 195 2OO
Thir Ser Wall Pro Wall Pro Pro Ala Ala Ala Pro Pro Pro Pro Ala Thir 21 O 215 22O
Pro Ile Wall Pro Wall Wall Pro Pro Thir Pro Pro Wall Wall Lys 225 23 O 235 24 O
Gly Wall Arg Lys Ala Asp Thr Thir Thir Pro Thir Thir Ser Ala Ile 245 250 255
Thir Ala Ser Arg Ser Glu Ser Pro Pro Pro Luell Ser Asp Pro Glin 26 O 265 27 O
Ala Wall Wall Ala Arg Arg Glu Ser Gly Gly Arg Pro Ile Pro 27s 28O 285
Pro Lys Asp Lieu. Glu Asp Gly Glu Wall Pro Glin His Ala Gly 29 O 295 3 OO
Lys Gly Luell Ser Glu. His Lieu. Arg Cys Asp Ser Ile Luell Arg 3. OS 310 315 32O US 9,301.962 B2 141 142 - Continued
Glu Met Luell Ser Lys His Ala Ala Tyr Ala Trp Pro Phe Tyr 3.25 330 335
Pro Wall Asp Ala Glu Ala Lell Glu Luell His Asp His Asp Ile Ile 34 O 345 35. O
His Pro Met Asp Lell Ser Thir Wall Lys Arg Met Asp Gly Arg 355 360 365
Glu Tyr Pro Asp Ala Glin Gly Phe Ala Ala Asp Wall Arg Luell Met Phe 37 O 375
Ser Asn Lys Tyr Asn Pro Pro Asp His Glu Wall Wall Ala Met 385 390 395 4 OO
Ala Arg Luell Glin Asp Wall Phe Glu Met Arg Phe Ala Met Pro 4 OS 415
Asp Glu Pro Wall Glu Ala Pro Ala Luell Pro Ala Pro Ala Ala Pro Met 425 43 O
Wall Ser Lys Gly Ala Glu Ser Ser Arg Ser Ser Glu Glu Ser Ser Ser 435 44 O 445
Asp Ser Gly Ser Ser Asp Ser Glu Glu Glu Arg Ala Thir Arg Luell Ala 450 45.5 460
Glu Luell Glin Glu Glin Lell Ala Wall His Glu Glin Lell Ala Ala Luell 465 470
Ser Glin Ala Pro Wall Asn Pro Lys Glu Lys Glu 485 490 495
Glu Lys Lys Glu Glu Glu Lys His SOO 505 51O
Wall Ala Glu Glu Lys Ala Wall Ala Pro Pro Ala 515 52O 525
Glin Ala Glin Glin Lys Ala Pro Ala Ala Asn Ser Thir Thir 53 O 535 54 O
Thir Ala Gly Arg Glin Lell Gly Gly Lys Glin Ala Ser Ala Ser 5.45 550 555 560
Asp Ser Glu Glu Glu Glu Glu Gly Luell Pro Met Ser Asp Glu 565 st O sts
Arg Glin Luell Ser Lell Asp Ile Asn Arg Luell Pro Gly Glu Luell 585 59 O
Gly Arg Wall Wall His Ile Ile Glin Ser Arg Glu Pro Ser Luell Arg Asp 595 605
Ser Asn Pro Asp Glu Ile Glu Ile Asp Phe Glu Thir Lell Pro Thir 610 615
Thir Luell Arg Glu Lell Glu Arg Wall Ser Lell Glin Lys 625 630 635 64 O
Glin Arg Pro Phe Ser Ala Ser Gly Lys Glin Ala Ala Lys Ser 645 650 655
Glu Glu Luell Ala Glin Glu Lys Glu Lell Glu Lys Arg Luell 660 665 67 O
Glin Asp Wall Ser Gly Glin Lell Ser Ser Ser Pro Ala Arg 675 68O 685
Glu Lys Pro Gly Ser Ala Pro Ser Gly Gly Pro Ser Arg Luell Ser Ser 69 O. 695 7 OO
Ser Ser Ser Ser Glu Ser Gly Ser Ser Ser Ser Ser Gly Ser Ser Ser 7 Os 71s 72O
Asp Ser Ser Asp Ser Glu 72 US 9,301.962 B2 143 144 - Continued
<210s, SEQ ID NO 3 &211s LENGTH: 722 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 3 Met Ser Ala Glu Ser Gly Pro Gly Thr Arg Lieu. Arg Asn Lieu Pro Val 1. 5 1O 15 Met Gly Asp Gly Lieu. Glu Thir Ser Gln Met Ser Thr Thr Glin Ala Glin 2O 25 3O
Ala Glin Pro Glin Pro Ala Asn Ala Ala Ser Thir Asn. Pro Pro Pro Pro 35 4 O 45 Glu Thir Ser Asn Pro Asn Llys Pro Lys Arg Glin Thr Asn Glin Lieu. Glin SO 55 6 O Tyr Lieu. Lieu. Arg Val Val Lieu Lys Thr Lieu. Trp Llys His Glin Phe Ala 65 70 7s 8O Trp Pro Phe Glin Glin Pro Val Asp Ala Wall Lys Lieu. Asn Lieu Pro Asp 85 90 95 Tyr Tyr Lys Ile Ile Llys Thr Pro Met Asp Met Gly Thr Ile Llys Lys 1OO 105 11 O Arg Lieu. Glu Asn. Asn Tyr Tyr Trp Asn Ala Glin Glu. Cys Ile Glin Asp 115 12 O 125 Phe Asn Thr Met Phe Thr Asn Cys Tyr Ile Tyr Asn Llys Pro Gly Asp 13 O 135 14 O Asp Ile Val Lieu Met Ala Glu Ala Lieu. Glu Lys Lieu. Phe Lieu Gln Lys 145 150 155 160
Ile Asn. Glu Lieu. Pro Thr Glu Glu Thir Glu Ile Met Ile Wall Glin Ala 1.65 17O 17s Lys Gly Arg Gly Arg Gly Arg Lys Glu Thr Gly Thr Ala Lys Pro Gly 18O 185 19 O
Wall Ser Thir Wall Pro Asn. Thir Thr Glin Ala Ser Thr Pro Pro Glin. Thir 195 2OO 2O5
Glin Thr Pro Glin Pro ASn Pro Pro Pro Wall Glin Ala Thr Pro His Pro 21 O 215 22O Phe Pro Ala Val Thr Pro Asp Lieu. Ile Val Glin Thr Pro Val Met Thr 225 23 O 235 24 O
Wall Wall Pro Pro Glin Pro Leul Glin. Thir Pro Pro Pro Wall Pro Pro Glin 245 250 255
Pro Glin Pro Pro Pro Ala Pro Ala Pro Glin Pro Wall Glin Ser His Pro 26 O 265 27 O Pro Ile Ile Ala Ala Thr Pro Gln Pro Val Lys Thr Lys Lys Gly Val 27s 28O 285 Lys Arg Lys Ala Asp Thir Thr Thr Pro Thr Thr Ile Asp Pro Ile His 29 O 295 3 OO Glu Pro Pro Ser Leu Pro Pro Glu Pro Llys Thir Thr Lys Lieu. Gly Glin 3. OS 310 315 32O
Arg Arg Glu Ser Ser Arg Pro Val Llys Pro Pro Llys Lys Asp Val Pro 3.25 330 335 Asp Ser Glin Gln His Pro Ala Pro Glu Lys Ser Ser Llys Val Ser Glu 34 O 345 35. O
Glin Lieu Lys Cys Cys Ser Gly Ile Lieu Lys Glu Met Phe Ala Lys Llys 355 360 365 His Ala Ala Tyr Ala Trp Pro Phe Tyr Llys Pro Val Asp Val Glu Ala 37 O 375 38O US 9,301.962 B2 145 146 - Continued
Lell Gly Luell His Asp Tyr Asp Ile Ile Lys His Pro Met Asp Met 385 390 395 4 OO
Ser Thir Ile Ser Lell Glu Ala Arg Glu Tyr Arg Asp Ala Glin 4 OS 415
Glu Phe Gly Ala Asp Wall Arg Luell Met Phe Ser Asn Tyr 425 43 O
Asn Pro Pro Asp His Glu Wall Wall Ala Met Ala Arg Lys Luell Glin Asp 435 44 O 445
Wall Phe Glu Met Arg Phe Ala Met Pro Asp Glu Pro Glu Glu Pro 450 45.5 460
Wall Wall Ala Wall Ser Ser Pro Ala Wall Pro Pro Pro Thir Wall Wall 465 470
Ala Pro Pro Ser Ser Ser Asp Ser Ser Ser Asp Ser Ser Ser Asp Ser 485 490 495
Asp Ser Ser Thir Asp Asp Ser Glu Glu Glu Arg Ala Glin Arg Luell Ala SOO 505
Glu Luell Glin Glu Glin Lell Ala Wall His Glu Glin Lell Ala Ala Luell 515 525
Ser Glin Pro Glin Glin Asn Lys Pro Glu Asp 53 O 535 54 O
Glu Glu Lys His Arg Glu Glu Wall Glu Glu Asn 5.45 550 555 560
Ser Ala Glu Pro Pro Pro Thir Lys Asn 565 st O sts
Asn Ser Ser Asn Ser Asn Wall Ser Lys Glu Pro Ala Pro Met 58O 585 59 O
Ser Pro Pro Pro Thir Glu Ser Glu Glu Glu Asp 595 605
Pro Met Ser Glu Glu Lys Arg Glin Luell Ser Lell Asp Ile Asn 610 615
Lell Pro Gly Glu Lys Lell Gly Arg Wall Wall His Ile Ile Glin Ser Arg 625 630 635 64 O
Glu Pro Ser Luell Lys Asn Ser Asn Pro Asp Glu Ile Glu Ile Asp Phe 645 650 655
Glu Thir Luell Lys Pro Ser Thir Luell Arg Glu Luell Glu Arg Tyr Wall Thir 660 665 67 O
Ser Luell Arg Lys Arg Lys Pro Glin Ala Glu Lys Wall Asp Wall 675 685
Ile Ala Gly Ser Ser Met Gly Phe Ser Ser Ser Glu Ser Glu 69 O. 695 7 OO
Ser Ser Ser Glu Ser Ser Ser Ser Asp Ser Glu Asp Ser Glu Thir Gly 7 Os 71O 71s 72O
Pro Ala
<210s, SEQ ID NO 4 &211s LENGTH: 947 212. TYPE : PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 4. Met Ser Leu Pro Ser Arg Glin Thr Ala Ile Ile Val Asn Pro Pro Pro 1. 5 15
Pro Glu Tyr Ile Asn. Thir Lys Lys Asn Gly Arg Lieu. Thir Asn Glin Lieu. 25 US 9,301.962 B2 147 148 - Continued
Glin Luell Glin Wall Wall Luell Asp Luell Trp Lys His Ser Phe 35 4 O 45
Ser Pro Phe Glin Arg Pro Wall Asp Ala Wall Lys Lell Glin Luell Pro 55 6 O
Asp Thir Ile Ile Asn Pro Met Asp Lell Asn Thir Ile Lys 65 70
Arg Luell Glu Asn Ala Lys Ala Ser Glu Ile Glu 85 90 95
Asp Phe Asn Thir Met Phe Ser Asn Cys Luell Tyr Asn Lys Pro Gly 105 11 O
Asp Asp Ile Wall Lell Met Ala Glin Ala Luell Glu Lell Phe Met Glin 115 12 O 125
Luell Ser Glin Met Pro Glin Glu Glu Glin Wall Wall Gly Wall Glu 13 O 135 14 O
Arg Ile Gly Thir Glin Glin Asn Ile Ala Wall Ser Ser Ala Lys 145 150 155 160
Glu Ser Ser Pro Ser Ala Thir Glu Lys Wall Phe Glin Glin Glu 1.65 17O 17s
Ile Pro Ser Wall Phe Pro Thir Ser Ile Ser Pro Lell Asn Wall Wall 18O 185 19 O
Glin Gly Ala Ser Wall Asn Ser Ser Ser Glin Thir Ala Ala Glin Wall Thir 195
Gly Wall Ala Asp Thir Thir Thir Pro Ala Thir Ser Ala 21 O 215 22O
Wall Ala Ser Ser Glu Phe Ser Pro Thr Phe Thr Glu Lys Ser Wall 225 23 O 235 24 O
Ala Luell Pro Pro Ile Glu Asn Met Pro Asn Wall Luell Pro Asp 245 250 255
Ser Glin Glin Glin Tyr Asn Wall Wall Lys Thir Wall Wall Thir Glu Glin 26 O 265 27 O
Lell Arg His Ser Glu Ile Luell Glu Met Lell Ala His 27s 285
Phe Ser Ala Trp Pro Phe Asn Pro Wall Asp Wall Asn Ala Luell 29 O 295 3 OO
Gly Luell His Asn Tyr Tyr Asp Wall Wall ASn Pro Met Asp Luell Gly 3. OS 310 315
Thir Ile Glu Lys Met Asp Asn Glin Glu Tyr Asp Ala Tyr Lys 3.25 330 335
Phe Ala Ala Asp Wall Arg Lell Met Phe Met ASn Lys Asn 34 O 345 35. O
Pro Pro Asp His Glu Wall Wall Thir Met Ala Arg Met Lell Glin Asp Wall 355 360 365
Phe Glu Thir His Phe Ser Lys Ile Pro Ile Glu Pro Wall Glu Ser Met 37 O 375
Pro Luell Ile Lys Thir Asp Ile Thir Glu Thir Thir Gly Arg Glu 385 390 395 4 OO
Asn Thir Asn Glu Ala Ser Ser Glu Gly Asn Ser Ser Asp Asp Ser Glu 4 OS 415
Asp Glu Arg Wall Lys Arg Lell Ala Lys Luell Glin Glu Glin Luell Ala 42O 425 43 O
Wall His Glin Glin Lell Glin Wall Luell Ser Glin Wall Pro Phe Arg Luell 435 44 O 445
Asn Glu Ser Glu Lys Glu Wall US 9,301.962 B2 149 150 - Continued
450 45.5 460
Asn Asn Ser Asn Glu Asn Pro Arg Met Cys Glu Glin Met Arg Luell 465 470 48O
Glu Ser Lys Arg Asn Glin Pro Lys Arg Glin Glin Phe 485 490 495
Ile Gly Luell Lys Ser Glu Asp Glu Asp Asn Ala Pro Met Asn SOO 505
Asp Glu Lys Arg Glin Lell Ser Luell Asn Ile ASn Lell Pro Gly Asp 515 525
Luell Gly Arg Wall Wall His Ile Ile Glin Ser Arg Glu Pro Ser Luell 53 O 535 54 O
Ser Asn Ser Asn Pro Asp Glu Ile Glu Ile Asp Phe Glu Thir Luell Lys 5.45 550 555 560
Ala Ser Thir Luell Arg Glu Lell Glu Tyr Wall Ser Ala Luell Arg 565 st O sts
Arg Pro Luell Lys Pro Pro Ala Lys Ile Met Met Ser Glu 585 59 O
Glu Luell His Ser Glin Glin Glu Luell Glu Arg Luell Luell Asp 595 605
Wall Asn Asn Glin Lell Asn Ser Arg Arg Glin Thir Ser Asp 610 615 62O
Thir Glin Pro Ser Lys Ala Wall Glu Asn Wall Ser Arg Lell Ser Glu Ser 625 630 635 64 O
Ser Ser Ser Ser Ser Ser Ser Ser Glu Ser Glu Ser Ser Ser Ser Asp 645 650 655
Lell Ser Ser Ser Asp Ser Ser Asp Ser Glu Ser Glu Met Phe Pro 660 665 67 O
Phe Thir Glu Wall Lys Pro Asn Asp Ser Pro Ser Glu Asn Wall 675 685
Met Asn Glu Ile Luell Pro Glu Gly Arg Thir Gly Wall Thir 69 O. 695 7 OO
Glin Ile Gly Cys Wall Glin Asp Thir Thir Ser Ala Asn Thir Thir Luell 7 Os
Wall His Glin Thir Thir Pro Ser His Wall Met Pro Pro Asn His His Glin 72 73 O 73
Lell Ala Phe Asn Tyr Glin Glu Luell Glu His Luell Glin Thir Wall Asn 740 74. 7 O
Ile Ser Pro Luell Glin Ile Lell Pro Pro Ser Gly Asp Ser Glu Glin Luell 760 765
Ser Asn Gly Ile Thir Wall Met His Pro Ser Gly Asp Ser Asp Thir Thir 770 775
Met Luell Glu Ser Glu Cys Glin Ala Pro Wall Glin Asp Ile Ile 78s 79 O 79.
Asn Ala Asp Ser Trp Ser Luell Gly Pro Wall Pro Ser 805 810 815
Gly Wall Met Lys Ser Ser Glu Luell Phe ASn Glin Phe Arg Ala 825 83 O
Ala Ile Glu Glu Wall Ala Arg Thir Glin Glu Lell Ile Arg 835 84 O 845
His Luell Glu Glin Asn Thir Lys Glu Luell Lys Ala Ser Glin Glu Asn Glin 850 855 860
Arg Asp Luell Gly Asn Gly Lell Thir Wall Glu Ser Phe Ser Asn Ile 865 87O 87s 88O US 9,301.962 B2 151 152 - Continued
Glin Asn Lys Cys Ser Gly Glu Glu Gln Lys Glu His Glin Glin Ser Ser 885 890 895 Glu Ala Glin Asp Llys Ser Llys Lieu. Trp Lieu. Lieu Lys Asp Arg Asp Lieu. 9 OO 905 91 O Ala Arg Gln Lys Glu Glin Glu Arg Arg Arg Arg Glu Ala Met Val Gly 915 92 O 925
Thir Ile Asp Met Thr Lieu. Glin Ser Asp Ile Met Thir Met Phe Glu Asn 93 O 935 94 O Asn Phe Asp 945
SEO ID NO 5 LENGTH: 25 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic primer
SEQUENCE: 5 tggaga agca tttgggitatic tactic 25
SEQ ID NO 6 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic primer SEQUENCE: 6 alagacggcat gct Caacaca
SEO ID NO 7 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic primer
SEQUENCE: 7 gagtgaggcc cagcatatgt C 21
SEQ ID NO 8 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic primer
SEQUENCE: 8 ccitctggatt ttctgagttg ca 22
SEO ID NO 9 LENGTH: 25 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic primer
SEQUENCE: 9 gctittgggac tocacaacta citatg 25
SEQ ID NO 10 LENGTH: 23 US 9,301.962 B2 153 154 - Continued
&212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer <4 OOs, SEQUENCE: 10 gattgtc.cat titt coccittg atc 23
<210s, SEQ ID NO 11 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer <4 OOs, SEQUENCE: 11 titt.ccc caat gctggttga 19
<210s, SEQ ID NO 12 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer <4 OOs, SEQUENCE: 12 aaccaaaatc cqttgcttico t 21
<210s, SEQ ID NO 13 &211s LENGTH: 2O & 212 TYPE DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer <4 OOs, SEQUENCE: 13 gctgattic ct gaggcc ctitt
<210s, SEQ ID NO 14 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer
<4 OOs, SEQUENCE: 14
Cagggcagca agggacat 18
<210s, SEQ ID NO 15 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer
<4 OOs, SEQUENCE: 15 cgc.caacaga gaaacaac at ttag 24
<210s, SEQ ID NO 16 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer
<4 OOs, SEQUENCE: 16 US 9,301.962 B2 155 156 - Continued c caaccagga titcggat.ctt t 21
<210s, SEQ ID NO 17 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer <4 OOs, SEQUENCE: 17 cctic cc gttt gttct caact td 22
<210s, SEQ ID NO 18 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer <4 OOs, SEQUENCE: 18 ggtgcatago citgttcctta ttg 23
<210s, SEQ ID NO 19 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer
<4 OOs, SEQUENCE: 19 tgcacagaat agcaagtic Catca 23
<210s, SEQ ID NO 2 O &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer
<4 OOs, SEQUENCE: 2O tgtggcgaga tigcticttgaa
What is claimed is: 45 R is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, 1. A method of reducing or inhibiting spermatogenesis in a haloalkyl, hydroxy, alkoxy, or —COO—Rs, each of healthy fertile male subject in need thereof, the method com which is optionally substituted; prising administering an amount of a compound represented ring A is aryl or heteroaryl; by structural formula (I) sufficient to induce at least one of each R is independently alkyl, cycloalkyl, heterocy aZoospermia, oligoZoospermia, or asthenoZoospermia: 50 cloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two R together with the atoms to which each is attached, can form a fused aryl or het (I) eroaryl group; R R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, -sN 55 each of which is optionally substituted; f M R R is —(CH)-L, in which n is 0-3 and L is —COO R. (R)-- A || —CO—Rs. —CO N(RR), —S(O) R. y ^ R2 —S(O), N(RR), N(RR), N(R)C(O)R option N ally substituted aryl, or optionally substituted het lsS \/ 60 eroaryl; RB X R is H. D. (deuterium), halogen, or optionally substituted alkyl: each R is independently selected from the group consist wherein ing of X is N or CRs: 65 (i) H, aryl, substituted aryl, heteroaryl, or substituted het Rs is H. alkyl, cycloalkyl, heterocycloalkyl, aryl, or het eroaryl; eroaryl, each of which is optionally substituted; (ii) heterocycloalkyl or substituted heterocycloalkyl: