# Patent

## Title
Male contraceptive compositions and methods of use

## Abstract

The invention relates to compositions and methods for effecting male contraception.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is the U.S. National Stage of International Application No. PCT/US2011/036667, filed May 16, 2011, which designates the U.S., published in English, and claims the benefit of U.S. Provisional Application No. 61/334,991, filed May 14, 2010, U.S. Provisional Application No. 61/370,745, filed Aug. 4, 2010, U.S. Provisional Application No. 61/375,863, filed Aug. 22, 2010, U.S. Provisional Application No. 61/467,376, filed Mar. 24, 2011, and U.S. Provisional Application No. 61/467,299, filed Mar. 24, 2011. The enire teachings of the above applications are incorporated herein by reference.
Incorporation By Reference Of Material In AscII Text File
This application incorporates by reference the Sequence Listing contained in the following ASCII text file being submitted concurrently herewith:

    
    
        a) File name: 48051003002sequence.txt; created Apr. 24, 2013, 30 KB in size.
    
    




STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
This work was supported by the following grant from the National Institutes of Health, Grant No: K08CA128972 (Bradner). The government has certain rights in the invention.


BACKGROUND OF THE INVENTION
Although  ̃4% of the mammalian genome encodes genes expressed in male germ cells during spermatogenesis, contraceptive drugs for men have remained elusive. To date, the only drugs in clinical trials are testosterone analogs that alter endogenous androgen production. This lack of contraceptive alternatives for men is partially responsible for the high rate of unplanned pregnancies, especially in teenagers, and the associated maternal mortality and ethical, social, and financial costs associated with abortions and deliveries to single mothers. To approach this dearth of contraceptive alternatives for men, it is desirable to develop small molecules that could target spermatogenic-specific proteins that have been shown to be essential for both spermatogenesis and fertility in mammals. One such contraceptive target is the bromodomain testis-specific protein, BRDT.
BRDT is a tissue-restricted, chromatin remodeling protein expressed in pachytene spermatocytes, diplotene spermatocytes, and round spermatids. During post-meiotic maturation, BRDT localizes to the nucleus and reorganizes hyperacetylated histones through twin acetyl-lysine recognition modules, or bromodomains. The essential role of BRDT in spermatogenesis is mediated by the first bromodomain (BRDT(1)), which binds the tetra-acetylated amino-terminal tail of histone 4 (H4Kac4) with moderate potency (20 μM). Structural studies of murine BRDT have demonstrated that BRDT(1) binds a diacetylated histone 4 peptide (H4K5ac8ac) in part through a conserved asparagine, akin to seminal studies of other bromodomain co-activator proteins. Genetic studies of BRDT have demonstrated that selective deletion of the BRDT(1)-encoding region is sufficient to confer sterility in homozygous male mice, and a recently published genome-wide association study of idiopathic male infertility identified single nucleotide polymorphisms of BRDT as significantly associated with oligozoospermia or azoospermia in European men. These insights establish a compelling rationale to target BRDT for a contraceptive effect.
SUMMARY OF THE INVENTION
As described below, this invention provides novel compounds and compositions for effecting male contraception. The invention also provides methods for using such compounds and compositions in a male subject.
In one aspect, the invention provides methods for reducing or inhibiting spermatogenesis in a male subject. In embodiments, the methods involve administering an effective amount of a compound or a salt thereof that inhibits a bromodomain testis-specific protein (BRDT) to the male subject.
In one aspect, the invention provides methods for reducing the rate of male fertility in a subject. In embodiments, the methods involve administering an effective amount of a compound or a salt thereof that inhibits a BRDT to the male subject.
In the above aspects, the methods involve administering the compound or a salt thereof in an amount sufficient to reduce sperm number and/or reduce sperm motility.
In the above aspects, the methods involve administering the compound or a salt thereof in an amount sufficient to induce azoospermia, oligozoospermia, and/or asthenozoospermia. In embodiments, the methods induce a contraceptive effect in the subject.
In aspect of the invention, the invention provides pharmaceutical compositions having a compound that inhibits BRDT or a pharmaceutically acceptable salt or prodrug thereof. In embodiments, the compound or a salt thereof is present in a amount effective to reduce or inhibit spermatogenesis in a male subject.
In embodiments, the compound or a salt thereof is present in an amount effective to reduce sperm number and/or reduce sperm motility.
In embodiments, the compound or a salt thereof is present in a amount effective to induce azoospermia, oligozoospermia, and/or asthenozoospermia. In related embodiments, the compound or a salt thereof is present in a amount effective to induce a contraceptive effect in the subject.
In any of the above aspects, the compound or a salt thereof is administered to the subject using any dosage and/or route of administration described herein. In embodiments, the compound or a salt thereof is administered to the subject orally, transdermally, or by injection. In related embodiments, the compound or a salt thereof is administered in the form of a tablet or capsule. In related embodiments, the compound or a salt thereof is administered by parenteral injection, intramuscular injection, intravenous injection, subcutaneous implantation, subcutaneous injection, or transdermal preparation.
In any of the above aspects, the compound or a salt thereof is administered in combination with a pharmaceutically acceptable carrier, excipient, or diluent.
In any of the above aspects, administration of the compound or a salt thereof reduces epididymal sperm number by at least about 25% of the sperm number present in a control. In embodiments, administration of the compound or a salt thereof reduces epididymal sperm number by at least about 10% of the sperm number present in a control. In related embodiments, only about 5% of the spermatozoa remaining show progressive motility after administration of the compound or a salt thereof.
In any of the above aspects, administration of the compound or a salt thereof lowers the spermatozoa concentration to not more than 3 million/mL, 2 million/mL, 1 million/mL, 0.5 million/mL, 0.25 million/mL, or 0.1 million/mL. In related embodiments, administration of the compound or a salt thereof lowers the spermatozoa concentration to not more than 0.1 million/mL.
In one aspect, the invention provides kits for reducing male fertility. In embodiments, the kits contain an effective amount of a compound that inhibits BRDT or a pharmaceutically acceptable salt or prodrug thereof. In embodiments, the kits contain instructions for using the inhibitor in any of the methods described herein.
In any of the above aspects, the compound is JQ1 or a compound of any of Formulas I-XXII, or any compound disclosed herein, or a derivative or salt thereof. In embodiments, the compound is JQ1 or a pharmaceutically acceptable salt or prodrug thereof.
In any of the above aspects, the compound or a salt thereof is administered in combination with at least one additional male contraceptive agent or device. In embodiments, the additional male contraceptive is a condom. In embodiments, the additional male contraceptive is a modulator of testosterone production, androgen receptor function or stability.
Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations disclosed herein, including those pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and, together with the description, serve to explain the principles of the invention.
Definitions
To facilitate an understanding of the present invention, a number of terms and phrases are defined below.
The term “reducing or inhibiting spermatozoa emission” refers to lowering the amount of spermatozoa present in seminal fluid during discharge of the seminal fluid from a male subject. Reduction or inhibition of spermatozoa levels in seminal fluid can be effected by suppressing spermatogenesis, inducing azoospermia, inducing oligozoospermia, and the like. Thus, in the context of the present invention, “reducing or inhibiting spermatozoa emission” has the effect of inhibiting and/or reducing the rate of fertilization when the discharged seminal fluid contacts ova from a female subject.
“Spermatogenesis” refers to the overall process of gametogenesis in the male. Spermatogenesis takes place in the seminiferous tubule and is directly regulated by levels of follicle stimulating hormone and androgen at the periphery of the seminiferous tubule, particularly upon the Sertoli cells.
The term “azoospermia” refers to a spermatozoa content below 1 million per mL seminal fluid, approaching levels of zero spermatozoa content, and are the result of suppression of spermatogenesis.
The term “oligozoospermia” refers to a spermatozoa content between 20 and one million per mL (mill/mL) seminal fluid, and are the result of inhibited levels of spermatogenesis.
By “bromodomain” is meant a portion of a polypeptide that recognizes acetylated lysine residues. In one embodiment, a bromodomain of a BET family member polypeptide comprises approximately 110 amino acids and shares a conserved fold comprising a left-handed bundle of four alpha helices linked by diverse loop regions that interact with chromatin.
By “BET family polypeptide” is meant a polypeptide comprising two bromodomains and an extraterminal (ET) domain or a fragment thereof having transcriptional regulatory activity or acetylated lysine binding activity. Exemplary BET family members include BRD2, BRD3, BRD4 and BRDT.
By “BRD2 polypeptide” is meant a protein or fragment thereof having at least 85% identity to NP_005095 that is capable of binding chromatin or regulating transcription.
The sequence (SEQ ID NO: 1) of an exemplary BRD2 polypeptide follows:








MLQNVTPHNKLPGEGNAGLLGLGPEAAAPGKRIRKPSLLYEGFESPTMASVPALQLTPA



 


NPPPPEVSNPKKPGRVTNQLQYLHKVVMKALWKHQFAWPFRQPVDAVKLGLPDYHKIIK


 


QPMDMGTIKRRLENNYYWAASECMQDFNTMFTNCYIYNKPTDDIVLMAQTLEKIFLQKV


 


ASMPQEEQELVVTIPKNSHKKGAKLAALQGSVTSAHQVPAVSSVSHTALYTPPPEIPTT


 


VLNIPHPSVISSPLLKSLHSAGPPLLAVTAAPPAQPLAKKKGVKRKADTTTPTPTAILA


 


PGSPASPPGSLEPKAARLPPMRRESGRPIKPPRKDLPDSQQQHQSSKKGKLSEQLKHCN


 


GILKELLSKKHAAYAWPFYKPVDASALGLHDYHDIIKHPMDLSTVKRKMENRDYRDAQE


 


FAADVRLMFSNCYKYNPPDHDVVAMARKLQDVFEFRYAKMPDEPLEPGPLPVSTAMPPG


 


LAKSSSESSSEESSSESSSEEEEEEDEEDEEEEESESSDSEEERAHRLAELQEQLRAVH


 


EQLAALSQGPISKPKRKREKKEKKKKRKAEKHRGRAGADEDDKGPRAPRPPQPKKSKKA


 


SGSGGGSAALGPSGFGPSGGSGTKLPKKATKTAPPALPTGYDSEEEEESRPMSYDEKRQ


 


LSLDINKLPGEKLGRVVHIIQAREPSLRDSNPEEIEIDFETLKPSTLRELERYVLSCLR


 


KKPRKPYTIKKPVGKTKEELALEKKRELEKRLQDVSGQLNSTKKPPKKANEKTESSSAQ


 


QVAVSRLSASSSSSDSSSSSSSSSSSDTSDSDSG






By “BRD2 nucleic acid molecule” is meant a polynucleotide encoding a BRD2 polypeptide or fragment thereof.
By “BRD3 polypeptide” is meant a protein or fragment thereof having at least 85% identity to NP_031397.1 that is capable of binding chromatin or regulating transcription.
The sequence (SEQ ID NO: 2) of an exemplary BRD3 polypeptide follows:








  1 mstattvapa gipatpgpvn ppppevsnps kpgrktnqlq ymqnvvvktl wkhqfawpfy



 


 61 qpvdaiklnl pdyhkiiknp mdmgtikkrl ennyywsase cmqdfntmft ncyiynkptd


 


121 divlmaqale kiflqkvaqm pqeevellpp apkgkgrkpa agaqsagtqq vaayssyspa


 


181 tpfqsvpptv sqtpviaatp vptitanvts vpvppaaapp ppatpivpvv pptppvvkkk


 


241 gvkrkadttt pttsaitasr sesppplsdp kqakvvarre sggrpikppk kdledgevpq


 


301 hagkkgklse hlrycdsilr emlskkhaay awpfykpvda ealelhdyhd iikhpmdlst


 


361 vkrkmdgrey pdaqgfaadv rlmfsncyky nppdhevvam arklqdvfem rfakmpdepv


 


421 eapalpapaa pmvskgaess rsseesssds gssdseeera trlaelqeql kavheqlaal


 


481 sqapvnkpkk kkekkekekk kkdkekekek hkvkaeeekk akvappakqa qqkkapakka


 


541 nstttagrql kkggkqasas ydseeeeegl pmsydekrql sldinrlpge klgrvvhiiq


 


601 srepslrdsn pdeieidfet lkpttlrele ryvksclqkk qrkpfsasgk kqaakskeel


 


661 aqekkkelek rlqdvsgqls sskkparkek pgsapsggps rlsssssses gsssssgsss


 


721 dssdse






By “Brd3 nucleic acid molecule” is meant a polynucleotide encoding a BRD3 polypeptide.
By “BRD4 polypeptide” is meant a protein or fragment thereof having at least 85% identity to NP_055114(SEQ ID NO: 3) that is capable of binding chromatin or regulating transcription.








  1 msaesgpgtr lrnlpvmgdg letsqmsttq aqaqpqpana astnppppet snpnkpkrqt



 


 61 nqlqyllrvv lktlwkhqfa wpfqqpvdav klnlpdyyki iktpmdmgti kkrlennyyw


 


121 naqeciqdfn tmftncyiyn kpgddivlma ealeklflqk inelpteete imivqakgrg


 


181 rgrketgtak pgvstvpntt qastppqtqt pqpnpppvqa tphpfpavtp dlivqtpvmt


 


241 vvppqplqtp ppvppqpqpp papapqpvqs hppiiaatpq pvktkkgvkr kadtttptti


 


301 dpiheppslp pepkttklgq rressrpvkp pkkdvpdsqq hpapeksskv seqlkccsgi


 


361 lkemfakkha ayawpfykpv dvealglhdy cdiikhpmdm stiksklear eyrdaqefga


 


421 dvrlmfsncy kynppdhevv amarklqdvf emrfakmpde peepvvayss pavppptkvv


 


481 appsssdsss dsssdsdsst ddseeeraqr laelqeqlka vheqlaalsq pqqnkpkkke


 


541 kdkkekkkek hkrkeeveen kkskakeppp kktkknnssn snvskkepap mkskppptye


 


601 seeedkckpm syeekrqlsl dinklpgekl grvvhiiqsr epslknsnpd eieidfetlk


 


661 pstlrelery vtsclrkkrk pqaekvdvia gsskmkgfss sesesssess ssdsedsetg


 


721 pa






By “Brd4 nucleic acid molecule” is meant a polynucleotide that encodes a BRD4 polypeptide.
By “BRDT polypeptide is meant a protein or fragment thereof having at least 85% identity to NP_001717 (SEQ ID NO: 4) that is capable of binding chromatin or regulating transcription.








  1 mslpsrqtai ivnppppeyi ntkkngrltn qlqylqkvvl kdlwkhsfsw pfqrpvdavk



 


 61 lqlpdyytii knpmdlntik krlenkyyak aseciedfnt mfsncylynk pgddivlmaq


 


121 aleklfmqkl sqmpqeeqvv gvkerikkgt qqniayssak eksspsatek vfkqqeipsv


 


181 fpktsispln vvqgasvnss sqtaaqvtkg vkrkadtttp atsavkasse fsptfteksv


 


241 alppikenmp knvlpdsqqq ynvvktvkvt eqlrhcseil kemlakkhfs yawpfynpvd


 


301 vnalglhnyy dvvknpmdlg tikekmdnqe ykdaykfaad vrlmfmncyk ynppdhevvt


 


361 marmlqdvfe thfskipiep vesmplcyik tditettgre ntneassegn ssddsederv


 


421 krlaklqeql kavhqqlqvl sqvpfrklnk kkekskkekk kekvnnsnen prkmceqmrl


 


481 kekskrnqpk krkqqfiglk sedednakpm nydekrqls1 ninklpgdkl grvvhiiqsr


 


541 epslsnsnpd eieidfetlk astlreleky vsaclrkrpl kppakkimms keelhsqkkq


 


601 elekrlldvn nqlnsrkrqt ksdktqpska venvsrlses sssssssses essssdlsss


 


661 dssdsesemf pkftevkpnd spskenvkkm knecilpegr tgvtqigycv qdttsanttl


 


721 vhqttpshvm ppnhhqlafn yqelehlqtv knisplqilp psgdseqlsn gitvmhpsgd


 


781 sdttmlesec qapvqkdiki knadswkslg kpvkpsgvmk ssdelfnqfr kaaiekevka


 


841 rtqelirkhl eqntkelkas qenqrdlgng ltvesfsnki qnkcsgeeqk ehqqsseaqd


 


901 ksklwllkdr dlarqkeqer rrreamvgti dmtlqsdimt mfennfd






By “BRDT nucleic acid molecule” is meant a polynucleotide encoding a BRDT polypeptide.
“Administering” is defined herein as a means of providing an agent to a subject in a manner that results in the agent being inside the subject's body. Such an administration can be by any route including, without limitation, oral, transdermal, mucosal (e.g., vagina, rectum, oral, or nasal mucosa), by injection (e.g., subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal), or by inhalation (e.g., oral or nasal). Pharmaceutical preparations are given by forms suitable for the desired route of administration.
By “agent” or “compound” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
By “alteration” is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.
By “analog” is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains at least some of the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.
As used herein, the term “alkyl” means a saturated straight chain or branched non-cyclic hydrocarbon typically having from 1 to 10 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylpentyl, 2,2-dimethylhexyl, 3,3-dimethylpentyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl, 3,3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl and the like. Alkyl groups included in compounds of this invention may be unsubstituted, or optionally substituted with one or more substituents, such as amino, alkylamino, arylamino, heteroarylamino, alkoxy, alkylthio, oxo, halo, acyl, nitro, hydroxyl, cyano, aryl, heteroaryl, alkylaryl, alkylheteroaryl, aryloxy, heteroaryloxy, arylthio, heteroarylthio, arylamino, heteroarylamino, carbocyclyl, carbocyclyloxy, carbocyclylthio, carbocyclylamino, heterocyclyl, heterocyclyloxy, heterocyclylamino, heterocyclylthio, and the like. Lower alkyls are typically preferred for the compounds of this invention.
As used herein, the term an “aromatic ring” or “aryl” means a monocyclic or polycyclic-aromatic ring or ring radical comprising carbon and hydrogen atoms. Examples of suitable aryl groups include, but are not limited to, phenyl, tolyl, anthacenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl. An aryl group can be unsubstituted or optionally is substituted with one or more substituents, e.g., substituents as described herein for alkyl groups (including without limitation alkyl (preferably, lower alkyl or alkyl substituted with one or more halo), hydroxy, alkoxy (preferably, lower alkoxy), alkylthio, cyano, halo, amino, boronic acid (—B(OH)2, and nitro). In certain embodiments, the aryl group is a monocyclic ring, wherein the ring comprises 6 carbon atoms.
The term “diastereomers” refers to stereoisomers with two or more centers of dissymmetry and whose molecules are not minor images of one another.
The term “enantiomers” refers to two stereoisomers of a compound which are non-superimposable minor images of one another. An equimolar mixture of two enantiomers is called a “racemic mixture” or a “racemate.”
The term “halogen” designates —F, —Cl, —Br or —I.
The term “haloalkyl” is intended to include alkyl groups as defined above that are mono-, di- or polysubstituted by halogen, e.g., fluoromethyl and trifluoromethyl.
The term “hydroxyl” means —OH.
The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-4 ring heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S, and the remainder ring atoms being carbon. Heteroaryl groups may be optionally substituted with one or more substituents, e.g., substituents as described herein for aryl groups. Examples of heteroaryl groups include, but are not limited to, pyridyl, furanyl, benzodioxolyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl, thiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, triazolyl, thiadiazolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxadiazolyl, and indolyl.
The term “heterocyclic” as used herein, refers to organic compounds that contain at least at least one atom other than carbon (e.g., S, O, N) within a ring structure. The ring structure in these organic compounds can be either aromatic or, in certain embodiments, non-aromatic. Some examples of heterocyclic moeities include, are not limited to, pyridine, pyrimidine, pyrrolidine, furan, tetrahydrofuran, tetrahydrothiophene, and dioxane.
The term “isomers” or “stereoisomers” refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
The term “isotopic derivatives” includes derivatives of compounds in which one or more atoms in the compounds are replaced with corresponding isotopes of the atoms. For example, an isotopic derivative of a compound containg a carbon atom (C12) would be one in which the carbon atom of the compound is replaced with the C13 isotope.
By “computer modeling” is meant the application of a computational program to determine one or more of the following: the location and binding proximity of a ligand to a binding moiety, the occupied space of a bound ligand, the amount of complementary contact surface between a binding moiety and a ligand, the deformation energy of binding of a given ligand to a binding moiety, and some estimate of hydrogen bonding strength, van der Waals interaction, hydrophobic interaction, and/or electrostatic interaction energies between ligand and binding moiety. Computer modeling can also provide comparisons between the features of a model system and a candidate compound. For example, a computer modeling experiment can compare a pharmacophore model of the invention with a candidate compound to assess the fit of the candidate compound with the model.
By a “computer system” is meant the hardware means, software means and data storage means used to analyse atomic coordinate data. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means and data storage means. Desirably a monitor is provided to visualise structure data. The data storage means may be RAM or means for accessing computer readable media of the invention. Examples of such systems are microcomputer workstations available from Silicon Graphics Incorporated and Sun Microsystems running Unix based, Windows NT or IBM OS/2 operating systems.
By “computer readable media” is meant any media which can be read and accessed directly by a computer e.g. so that the media is suitable for use in the above-mentioned computer system. The media include, but are not limited to: magnetic storage media such as floppy discs, hard disc storage medium and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.
By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
By “effective amount” is meant the amount of an agent required to provide contraception to an otherwise fertile male. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.
The term “enantiomers” refers to two stereoisomers of a compound which are non-superimposable minor images of one another. An equimolar mixture of two enantiomers is called a “racemic mixture” or a “racemate.”
The term “halogen” designates —F, —Cl, —Br or —I.
The term “haloalkyl” is intended to include alkyl groups as defined above that are mono-, di- or polysubstituted by halogen, e.g., fluoromethyl and trifluoromethyl.
The term “hydroxyl” means —OH.
The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-4 ring heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S, and the remainder ring atoms being carbon. Heteroaryl groups may be optionally substituted with one or more substituents. Examples of heteroaryl groups include, but are not limited to, pyridyl, furanyl, benzodioxolyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl thiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, triazolyl, thiadiazolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxadiazolyl, and indolyl.
The term “heterocyclic” as used herein, refers to organic compounds that contain at least at least one atom other than carbon (e.g., S, O, N) within a ring structure. The ring structure in these organic compounds can be either aromatic or non-aromatic. Some examples of heterocyclic moeities include, are not limited to, pyridine, pyrimidine, pyrrolidine, furan, tetrahydrofuran, tetrahydrothiophene, and dioxane.
The term “isomers” or “stereoisomers” refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
The term “isotopic derivatives” includes derivatives of compounds in which one or more atoms in the compounds are replaced with corresponding isotopes of the atoms. For example, an isotopic derivative of a compound containing a carbon atom (C12) would be one in which the carbon atom of the compound is replaced with the C13 isotope.
The invention provides a number of targets that are useful for the development of highly specific drugs to reduce fertility in a male subject. In addition, the methods of the invention provide a facile means to identify other contraceptive therapies that are safe for use in male subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.
By “fitting” is meant determining by automatic, or semi-automatic means, interactions between one or more atoms of an agent molecule and one or more atoms or binding sites of a BET family member (e.g., a bromodomain of BRD2, BRD3, BRD4 and BRDT), and determining the extent to which such interactions are stable. Various computer-based methods for fitting are described further herein.
By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
“Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
The term “obtaining” as in “obtaining compound” is intended to include purchasing, synthesizing or otherwise acquiring the compound.
The term “optical isomers” as used herein includes molecules, also known as chiral molecules, that are exact non-superimposable mirror images of one another.
The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
“Pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.
“Pharmaceutically acceptable excipient, carrier or adjuvant” refers to an excipient, carrier or adjuvant that can be administered to a subject, together with an agent, e.g., any of the compounds described herein, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.
The terms “polycyclyl” or “polycyclic radical” refer to the radical of two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are “fused rings”. Rings that are joined through non-adjacent atoms are termed “bridged” rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.
The term “polymorph” as used herein, refers to solid crystalline forms of a compound of the present invention or complex thereof. Different polymorphs of the same compound can exhibit different physical, chemical and/or spectroscopic properties. Different physical properties include, but are not limited to stability (e.g., to heat or light), compressibility and density (important in formulation and product manufacturing), and dissolution rates (which can affect bioavailability). Differences in stability can result from changes in chemical reactivity (e.g., differential oxidation, such that a dosage form discolors more rapidly when comprised of one polymorph than when comprised of another polymorph) or mechanical characteristics (e.g., tablets crumble on storage as a kinetically favored polymorph converts to thermodynamically more stable polymorph) or both (e.g., tablets of one polymorph are more susceptible to breakdown at high humidity). Different physical properties of polymorphs can affect their processing.
The term “prodrug” includes compounds with moieties which can be metabolized in vivo. Generally, the prodrugs are metabolized in vivo by esterases or by other mechanisms to active drugs. Examples of prodrugs and their uses are well-known in the art (See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19). The prodrugs can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Hydroxyl groups can be converted into esters via treatment with a carboxylic acid. Examples of prodrug moieties include substituted and unsubstituted, branch or unbranched lower alkyl ester moieties, (e.g., propionoic acid esters), lower alkenyl esters, di-lower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethyl ester), acylamino lower alkyl esters (e.g., acetyloxymethyl ester), acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g., with methyl, halo, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower-alkyl amides, di-lower alkyl amides, and hydroxy amides. Preferred prodrug moieties are propionoic acid esters and acyl esters. Prodrugs which are converted to active forms through other mechanisms in vivo are also included.
Furthermore the indication of stereochemistry across a carbon-carbon double bond is also opposite from the general chemical field in that “Z” refers to what is often referred to as a “cis” (same side) conformation whereas “E” refers to what is often referred to as a “trans” (opposite side) conformation. Both configurations, cis/trans and/or Z/E are encompassed by the compounds of the present invention.
With respect to the nomenclature of a chiral center, the terms “d” and “1” configuration are as defined by the IUPAC Recommendations. As to the use of the terms, diastereomer, racemate, epimer and enantiomer, these will be used in their normal context to describe the stereochemistry of preparations.
By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
By “reference” is meant a standard or control condition.
A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well-known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well-known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 85% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 85%, 90%, 95%, 99% or even 100% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e.sup.-3 and e.sup.-100 indicating a closely related sequence.
By “reduces” or “increases” is meant a negative or positive alteration, respectively, of at least about 10%, 25%, 50%, 75%, or 100% relative to a reference.
By “root mean square deviation” is meant the square root of the arithmetic mean of the squares of the deviations from the mean.
By “reducing cell survival” is meant to inhibit the viability of a cell or to induce cell death relative to a reference cell.
By “reference” is meant a standard or control condition.
A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
The term “sulfhydryl” or “thiol” means —SH.
As used herein, the term “tautomers” refers to isomers of organic molecules that readily interconvert by tautomerization, in which a hydrogen atom or proton migrates in the reaction, accompanied in some occasions by a switch of a single bond and an adjacent double bond.
As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
“An effective amount” refers to an amount of a compound, which confers a contraceptive effect on the treated subject. The effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). An effective amount of a compound described herein may range from about 1 mg/Kg to about 5000 mg/Kg body weight. Effective doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents. In embodiments of the present evention, “an effective amount” of an agent or composition is an amount sufficient to effect contraception.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.
The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to.”
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compounds, compositions, or methods provided herein can be combined with one or more of any of the other compounds, compositions, and methods provided herein.



DESCRIPTION OF THE DRAWINGS
FIG. 1 is a sequence comparison of human BRDT(1) with human BRD4(1) and mouse BRDT(1). Protein sequence alignment reveals a high degree of sequence identity between homologous and orthologous domains. Identical (red) and similar (yellow) residues are highlighted. Depicted above the residue sequences are schematic representations of major helical elements. Contacts between (+)-JQ1 and BRDT(1) are depicted with a black star. The conserved asparagine mediating acetyl-lysine recognition is depicted with a blue star.
FIGS. 2A-2C show BRDT inhibition by (+)-JQ1. FIG. 2A is the structure of the active (+)-JQ1 enantiomer. FIG. 2B is a plot showing the competitive inhibition of BRDT binding to synthetic biotinylated H4Kac4 by (+)-JQ1 (IC50: 11 nM) using a proximity detection assay. FIG. 2C includes a graph showing the results of the assay with 500 nM of the indicated compound. Error bars show standard deviation.
FIG. 3 is a MAFFT alignment of human BRDT and human BRD4.
FIG. 4 is a MAFFT alignment of human BRDT and mouse BRDT.
FIGS. 5A-5H show gross and histological analysis of testes from mice treated with JQ1 or vehicle control. FIG. 5A includes an image providing a gross analysis of testes from 9-week old mice injected with control or JQ1. FIG. 5B includes a graphical representation of testes weights (mg) from mice treated with control or JQ1 for 3-6 weeks, 6-9 weeks, or 6-12 weeks of age. Data represent the mean±standard error of the mean (SEM), and are annotated with P-values as obtained from a two-tailed t-test (* indicates significant at P<0.05). FIGS. 5C-5F include histological stains showing the histology of testes of 6-week old mice treated with (FIG. 5C) control or (FIG. 5D) JQ1 from 3-6 weeks of age, and 12-week old mice treated with (FIG. 5E) control or (FIG. 5F) JQ1 from 6-12 weeks of age. Intertubular islands of Leydig cells are depicted with arrows in FIGS. 5C-5F. Sertoli cell vacuolization (V) is highlighted in several tubules in FIG. 5F. FIGS. 5G and 5H include histological stains showing the histology of the epididymides from males treated with (FIG. 5G) control or (FIG. 5H) JQ1 from 6-12 weeks of age. Fewer spermatozoa and multiple large nucleated cells (black arrow) are observed in the epididymal lumen of the JQ1-treated mice (FIG. 5H) compared to the control epididymal lumen (FIG. 5G), which is densely packed with mature spermatozoa. FIGS. 5C-5H were photographed at the same magnification.
FIGS. 6A and 6B show epididymal sperm counts and fertilization potential. FIG. 6A is a graphical representation of the sperm counts obtained from the entire epididymides of males treated with JQ1 or control from 6-9 weeks of age or the tail (cauda) of the epididymis of males treated from 6-12 weeks of age. FIG. 6B includes a graph showing the in vitro developmental potential of oocytes obtained from superovulated females mated to males treated for 5 weeks with a control or JQ1. All data represent the mean±SEM, and are annotated with P-values as obtained from a two-tailed t-test (* indicates significant at P<0.05).
FIGS. 7A-7C show the molecular analysis of the testes of mice treated with JQ1 or control. FIG. 7A includes a graph showing the quantitative RT-PCR results for males treated from 6-12 weeks of age with JQ1 or a control solution. The mouse genes tested were Plzf (promyelocytic leukemia zinc-finger or Zbtb16), Stra8 (stimulated by retinoic acid gene 8), Brdt (bromodomain, testis-specific), Ccna1 (cyclin A1), Hist1 h1t (histone cluster 1, histone 1, testis-specific), Papolb (poly (A) polymerase beta or Tpap), Klf17 (Kruppel-like factor 17 or Zfp393), and Prm1 (protamine 1). Data represent the mean±SEM, and are annotated with P-values as obtained from a two-tailed t-test (* indicates significant at P<0.05; the P-value for Prm1 is 0.06). FIGS. 7B and 7C include immunohistochemical staining images of TNP2 in control-treated (FIG. 7B) and JQ1-treated (FIG. 7C) testes.
FIGS. 8A-8C show the effect of JQ1 on sperm count and testicular mass. In a repeat study, C57B6 mice were treated with JQ1 (50 mpk×8 weeks). FIG. 8A includes a graph showing sperm count in the test mice. FIG. 8B includes a graph showing testes weight in the test mice. FIG. 8C includes phase contrast images of sperm from test mice.
FIGS. 9A-9C show that the effects of JQ1 are reversed upon removal. FIG. 9A includes a graph showing sperm motility levels in test mice two months and four months after termination of JQ1 treatment. FIG. 9B includes a graph showing testes weight in test mice two months and four months after termination of JQ1 treatment. FIG. 9C includes a graph showing the sperm counts in test mice two months and four months after termination of JQ1 treatment.



DETAILED DESCRIPTION OF THE INVENTION
This invention is based, at least in part, on the discovery that a small-molecule inhibitor (JQ1) of the bromodomain and extra-terminal (BET) subfamily of epigenetic reader proteins is essential for chromatin remodeling during spermiogenesis. Biochemical analysis confirms that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. The invention is also based on the discovery that treatment of mice with JQ1 reduced the number and motility of spermatozoa, as well as testis size. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte stage cause a dramatic decrease in fertilized oocytes and a reversible contraceptive effect in males. Accordingly, the present invention is directed to a novel type of male contraceptive that can cross the blood:testis boundary and inhibits bromodomain activity during spermatogenesis.
Bromodomain-containing Proteins
Gene regulation is fundamentally governed by reversible, non-covalent assembly of macromolecules. Signal transduction to RNA polymerase requires higher-ordered protein complexes, spatially regulated by assembly factors capable of interpreting the post-translational modification states of chromatin. Epigenetic readers are structurally diverse proteins each possessing one or more evolutionarily conserved effector modules, which recognize covalent modifications of histone proteins or DNA. The ε-N-acetylation of lysine residues (Kac) on histone tails is associated with an open chromatin architecture and transcriptional activation3. Context-specific molecular recognition of acetyl-lysine is principally mediated by bromodomains.
Bromodomain-containing proteins are of substantial biological interest, as components of transcription factor complexes (TAF1, PCAF, GcnS and CBP) and determinants of epigenetic memory4. There are 41 human proteins containing a total of 57 diverse bromodomains. Despite large sequence variations, all bromodomains share a conserved fold comprising a left-handed bundle of four alpha helices (αZ, αA, αB, αC), linked by diverse loop regions (ZA and BC loops) that determine substrate specificity. Co-crystal structures with peptidic substrates showed that the acetyl-lysine is recognized by a central hydrophobic cavity and is anchored by a hydrogen bond with an asparagine residue present in most bromodomains5. The bromodomain and extra-terminal (BET)-family (BRD2, BRD3, BRD4 and BRDT) shares a common domain architecture comprising two N-terminal bromodomains that exhibit high level of sequence conservation, and a more divergent C-terminal recruitment domain6.
The invention features compositions and methods that are useful for inhibiting human bromodomain proteins.
Compounds of the Invention
The invention provides compounds (e.g., JQ1 and compounds of formulas delineated herein) that bind in the binding pocket of the apo crystal structure of the first bromodomain of a BET family member (e.g., BRDT, BRD2, BRD3, BRD4). Without wishing to be bound by theory, these compounds are particularly effective in reducing male fertility. In one approach, compounds useful for reducing male fertility are selected using a molecular docking program to identify compounds that are expected to bind to a bromodomain structural binding pocket. In certain embodiments, a compound of the invention can prevent, inhibit, or disrupt, or reduce by at least 10%, 25%, 50%, 75%, or 100% the biological activity of a BET family member (e.g., BRD2, BRD3, BRD4, BRDT) and/or disrupt the subcellular localization of such proteins, e.g., by binding to a binding site in a bromodomain apo binding pocket.
In certain embodiments, a compound of the invention is a small molecule having a molecular weight less than about 1000 daltons, less than 800, less than 600, less than 500, less than 400, or less than about 300 daltons. Examples of compounds of the invention include JQ1 and other compounds that bind the binding pocket of the apo crystal structure of the first bromodomain of a BET family member (e.g., BRD4 (hereafter referred to as BRD4(1); PDB ID 20SS). JQ1 is a novel thieno-triazolo-1,4-diazepine. The invention further provides pharmaceutically acceptable salts of such compounds.
In one aspect, the compound is a compound of Formula I:




wherein

    
    
        X is N or CR5;
        
            R5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
            RB is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R3, each of which is optionally substituted;
        
        
        ring A is aryl or heteroaryl;
        
            each RA is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two RA together with the atoms to which each is attached, can form a fused aryl or heteroaryl group;
        
        
        R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; each of which is optionally substituted;
        R1 is —(CH2)n-L, in which n is 0-3 and L is H, —COO—R3, —CO—R3, —CO—N(R3R4), —S(O)2—R3, —S(O)2—N(R3R4), N(R3R4), N(R4)C(O)R3, optionally substituted aryl, or optionally substituted heteroaryl;
        R2 is H, D (deuterium), halogen, or optionally substituted alkyl;
        each R3 is independently selected from the group consisting of:
        
            (i) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
            (ii) heterocycloalkyl or substituted heterocycloalkyl;
            (iii) —C1-C8 alkyl, —C2-C8 alkenyl or —C2-C8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C3-C12 cycloalkyl, substituted —C3-C12 cycloalkyl, —C3-C12 cycloalkenyl, or substituted —C3-C12 cycloalkenyl, each of which may be optionally substituted; and
            (iv) NH2, N═CR4R6;
            each R4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
            or R3 and R4 are taken together with the nitrogen atom to which they are attached to form a 4-10-membered ring;
            R6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R4 and R6 are taken together with the carbon atom to which they are attached to form a 4-10-membered ring;
        
        
        m is 0, 1, 2, or 3;
        provided that
        
            (a) if ring A is thienyl, X is N, R is phenyl or substituted phenyl, R2 is H, RB is methyl, and R1 is —(CH2)n-L, in which n is 1 and L is —CO—N(R3R4), then R3 and R4 are not taken together with the nitrogen atom to which they are attached to form a morpholino ring;
            (b) if ring A is thienyl, X is N, R is substituted phenyl, R2 is H, RB is methyl, and R1 is —(CH2)n-L, in which n is 1 and L is —CO—N(R3R4), and one of R3 and R4 is H, then the other of R3 and R4 is not methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl; and
            (c) if ring A is thienyl, X is N, R is substituted phenyl, R2 is H, RB is methyl, and R1 is —(CH2)n-L, in which n is 1 and L is —COO—R3, then R3 is not methyl or ethyl;
            or a salt, solvate or hydrate thereof.
        
        
    
    


In certain embodiments, R is aryl or heteroaryl, each of which is optionally substituted.
In certain embodiments, L is H, —COO—R3, —CO—N(R3R4), —S(O)2—R3, —S(O)2—N(R3R4), N(R3R4), N(R4)C(O)R3 or optionally substituted aryl. In certain embodiments, each R3 is independently selected from the group consisting of: H, —C1-C8 alkyl, containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; or NH2, N═CR4R6.
In certain embodiments, R2 is H, D, halogen or methyl.
In certain embodiments, RB is alkyl, hydroxyalkyl, haloalkyl, or alkoxy; each of which is optionally substituted.
In certain embodiments, RB is methyl, ethyl, hydroxy methyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, or COOCH2OC(O)CH3.
In certain embodiments, ring A is a 5 or 6-membered aryl or heteroaryl. In certain embodiments, ring A is thiofuranyl, phenyl, naphthyl, biphenyl, tetrahydronaphthyl, indanyl, pyridyl, furanyl, indolyl, pyrimidinyl, pyridizinyl, pyrazinyl, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, or 5,6,7,8-tetrahydroisoquinolinyl.
In certain embodiments, ring A is phenyl or thienyl.
In certain embodiments, m is 1 or 2, and at least one occurrence of RA is methyl.
In certain embodiments, each RA is independently H, an optionally substituted alkyl, or any two RA together with the atoms to which each is attached, can form an aryl.
In another aspect, the compound is a compound of Formula II:




wherein

    
    
        X is N or CR5;
        
            R5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
            RB is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R3, each of which is optionally substituted;
        
        
        each RA is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two RA together with the atoms to which each is attached, can form a fused aryl or heteroaryl group;
        R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
        R′1, is H, —COO—R3, —CO—R3, optionally substituted aryl, or optionally substituted heteroaryl;
        
            each R3 is independently selected from the group consisting of:
            (i) H, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
            (ii) heterocycloalkyl or substituted heterocycloalkyl;
            (iii) —C1-C8 alkyl, —C2-C8 alkenyl or —C2-C8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C3-C12 cycloalkyl, substituted —C3-C12 cycloalkyl; —C3-C12 cycloalkenyl, or substituted —C3-C12 cycloalkenyl; each of which may be optionally substituted;
        
        
        m is 0, 1, 2, or 3;
        provided that if R′1, is —COO—R3, X is N, R is substituted phenyl, and RB is methyl, then R3 is not methyl or ethyl;
        or a salt, solvate or hydrate thereof.
    
    


In certain embodiments, R is aryl or heteroaryl, each of which is optionally substituted. In certain embodiments, R is phenyl or pyridyl, each of which is optionally substituted. In certain embodiments, R is p-Cl-phenyl, o-Cl-phenyl, m-Cl-phenyl, p-F-phenyl, o-F-phenyl, m-F-phenyl or pyridinyl.
In certain embodiments, R′1, is —COO—R3, optionally substituted aryl, or optionally substituted heteroaryl; and R3 is —C1-C8 alkyl, which contains 0, 1, 2, or 3 heteroatoms selected from O, S, or N, and which may be optionally substituted. In certain embodiments, R′1, is —COO—R3, and R3 is methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, or t-butyl; or R′1, is H or optionally substituted phenyl.
In certain embodiments, RB is methyl, ethyl, hydroxy methyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, COOCH2OC(O)CH3.
In certain embodiments, RB is methyl, ethyl, hydroxy methyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, or COOCH2OC(O)CH3.
In certain embodiments, each RA is independently an optionally substituted alkyl, or any two RA together with the atoms to which each is attached, can form a fused aryl.
In certain embodiments, each RA is methyl.
In another aspect, the compound is a compound of formula III:




wherein

    
    
        X is N or CR5;
        
            R5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
            RB is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R3, each of which is optionally substituted;
        
        
        ring A is aryl or heteroaryl;
        
            each RA is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two RA together with the atoms to which each is attached, can form a fused aryl or heteroaryl group;
        
        
        R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
        each R3 is independently selected from the group consisting of:
        
            (i) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
            (ii) heterocycloalkyl or substituted heterocycloalkyl;
            (iii) —C1-C8 alkyl, —C2-C8 alkenyl or —C2-C8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C3-C12 cycloalkyl, substituted —C3-C12 cycloalkyl, —C3-C12 cycloalkenyl, or substituted —C3-C12 cycloalkenyl, each of which may be optionally substituted; and
            (iv) NH2, N═CR4R6;
            each R4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
            or R3 and R4 are taken together with the nitrogen atom to which they are attached to form a 4-10-membered ring;
            R6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R4 and R6 are taken together with the carbon atom to which they are attached to form a 4-10-membered ring;
        
        
        m is 0, 1, 2, or 3;
        provided that:
        
            (a) if ring A is thienyl, X is N, R is phenyl or substituted phenyl, RB is methyl, then R3 and R4 are not taken together with the nitrogen atom to which they are attached to form a morpholino ring; and
            (b) if ring A is thienyl, X is N, R is substituted phenyl, R2 is H, RB is methyl, and one of R3 and R4 is H, then the other of R3 and R4 is not methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl; and
        
        
        or a salt, solvate or hydrate thereof.
    
    


In certain embodiments, R is aryl or heteroaryl, each of which is optionally substituted. In certain embodiments, R is phenyl or pyridyl, each of which is optionally substituted.
In certain embodiments, R is p-Cl-phenyl, o-Cl-phenyl, m-Cl-phenyl, p-F-phenyl, o-F-phenyl, m-F-phenyl or pyridinyl. In certain embodiments, R3 is H, NH2, or N═CR4R6.
In certain embodiments, each R4 is independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl; each of which is optionally substituted.
In certain embodiments, R6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted.
In another aspect, the compound is a compound of formula IV:




wherein

    
    
        X is N or CR5;
        
            R5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
            RB is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R3, each of which is optionally substituted;
        
        
        ring A is aryl or heteroaryl;
        
            each RA is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two RA together with the atoms to which each is attached, can form a fused aryl or heteroaryl group;
        
        
        R1 is —(CH2)n-L, in which n is 0-3 and L is H, —COO—R3, —CO—R3, —CO—N(R3R4), —S(O)2—R3, —S(O)2—N(R3R4), N(R3R4), N(R4)C(O)R3, optionally substituted aryl, or optionally substituted heteroaryl;
        R2 is H, D, halogen, or optionally substituted alkyl;
        each R3 is independently selected from the group consisting of:
        
            (i) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
            (ii) heterocycloalkyl or substituted heterocycloalkyl;
            (iii) —C1-C8 alkyl, —C2-C8 alkenyl or —C2-C8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C3-C12 cycloalkyl, substituted —C3-C12 cycloalkyl, —C3-C12 cycloalkenyl, or substituted —C3-C12 cycloalkenyl, each of which may be optionally substituted; and
            (iv) NH2, N═CR4R6;
            each R4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
            or R3 and R4 are taken together with the nitrogen atom to which they are attached to form a 4-10-membered ring;
            R6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R4 and R6 are taken together with the carbon atom to which they are attached to form a 4-10-membered ring;
        
        
        m is 0, 1, 2, or 3;
        provided that
        
            (a) if ring A is thienyl, X is N, R2 is H, RB is methyl, and R1 is —(CH2)n-L, in which n is 0 and L is —CO—N(R3R4), then R3 and R4 are not taken together with the nitrogen atom to which they are attached to form a morpholino ring;
            (b) if ring A is thienyl, X is N, R2 is H, RB is methyl, and R1 is —(CH2)n-L, in which n is 0 and L is —CO—N(R3R4), and one of R3 and R4 is H, then the other of R3 and R4 is not methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl; and
            (c) if ring A is thienyl, X is N, R2 is H, RB is methyl, and R1 is —(CH2)n-L, in which n is 0 and L is —COO—R3, then R3 is not methyl or ethyl; or

a salt, solvate or hydrate thereof.

        
        
    
    


In certain embodiments, R1 is —(CH2)n-L, in which n is 0-3 and L is —COO—R3, optionally substituted aryl, or optionally substituted heteroaryl; and R3 is —C1-C8 alkyl, which contains 0, 1, 2, or 3 heteroatoms selected from O, S, or N, and which may be optionally substituted. In certain embodiments, n is 1 or 2 and L is alkyl or —COO—R3, and R3 is methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, or t-butyl; or n is 1 or 2 and L is H or optionally substituted phenyl.
In certain embodiments, R2 is H or methyl.
In certain embodiments, RB is methyl, ethyl, hydroxy methyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, COOCH2OC(O)CH3.
In certain embodiments, ring A is phenyl, naphthyl, biphenyl, tetrahydronaphthyl, indanyl, pyridyl, furanyl, indolyl, pyrimidinyl, pyridizinyl, pyrazinyl, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, or 5,6,7,8-tetrahydroisoquinolinyl.
In certain embodiments, each RA is independently an optionally substituted alkyl, or any two RA together with the atoms to which each is attached, can form an aryl.
The methods of the invention also relate to compounds of Formulae V-XXII, and to any compound described herein.
In another aspect, the compound is a compound represented by the formula:




or a salt, solvate or hydrate thereof.

In certain embodiments, the compound is (+)-JQ1:




or a salt, solvate or hydrate thereof.

In another aspect, the compound is a compound represented by the formula:




or a salt, solvate or hydrate thereof.

In another aspect, the compound is a compound represented by the formula:




or a salt, solvate or hydrate thereof.

In another aspect, the compound is a compound represented by any one of the following formulae:










or a salt, solvate or hydrate thereof.

In another aspect, the compound is a compound represented by any one of the following formulae:




or a salt, solvate or hydrate thereof.

In another aspect, the compound is a compound represented by any one of the following structures:




























or a salt, solvate or hydrate thereof.

In certain embodiments, a compound of the invention can be represented by one of the following structures:



















or a salt, solvate or hydrate thereof.

In one embodiment, the compound is represented by the structure:




or a salt, solvate or hydrate thereof.

In another embodiment, the compound is represented by the structure:




or a salt, solvate or hydrate thereof.

In another embodiment, the compound is represented by the structure:




or a salt, solvate or hydrate thereof.

In certain embodiments, a compound of the invention can have the opposite chirality of any compound shown herein.
In certain embodiments, the compound is a compound represented by Formula (V), (VI), or (VII):




in which R, R1, and R2 and RB have the same meaning as in Formula (I); Y is O, N, S, or CR5, in which R5 has the same meaning as in Formula (I); n is 0 or 1; and the dashed circle in Formula (VII) indicates an aromatic or non-aromatic ring; or a salt, solvate, or hydrate thereof.

In certain embodiments of any of the Formulae I-IV and VI (or any formula herein), R6 represents the non-carbonyl portion of an aldehyde shown in Table A, below (i.e., for an aldehyde of formula R6CHO, R6 is the non-carbonyl portion of the aldehyde). In certain embodiments, R4 and R6 together represent the non-carbonyl portion of a ketone shown in Table A (i.e., for a ketone of formula R6C(O)R4, R4 and R6 are the non-carbonyl portion of the ketone).













TABLE A








01
02
03
04
05
06










Plate 1













A























 


B


























 


C























 


D


























 


E


























 


F


























 


G


























 


H


























 







Plate 2













A























 


B


























 


C























 


D


























 


E


























 


F


























 


G


























 


H


























 







Plate 3













A























 


B


























 


C























 


D


























 


E


























 


F


























 


G


























 


H


























 







Plate 4













A























 


B


























 


C























 


D


























 


E


























 


F


























 


G


























 


H






07
08
09
10
11
12










Plate 1













A


























 


B


























 


C


























 


D























 


E


























 


F























 


G























 


H


























 







Plate 2













A


























 


B


























 


C


























 


D























 


E


























 


F























 


G























 


H


























 







Plate 3













A


























 


B


























 


C


























 


D























 


E


























 


F























 


G























 


H


























 







Plate 4













A


























 


B


























 


C


























 


D























 


E


























 


F























 


G




















 


H









In one embodiment, the compound is a compound is represented by the formula:




or a salt, solvate, or hydrate thereof.

In certain embodiments, the compound is (racemic) JQ1; in certain embodiments, the compound is (+)-JQ1. In certain embodiments, the compound is a compound selected from the group consisting of:




or a salt, solvate, or hydrate thereof.

Additional examples of compounds include compounds according to any of the follow formulae:













In Formulae IX-XXII, R and R′ can be, e.g., H, aryl, substituted aryl, heteroaryl, heteroaryl, heterocycloalkyl, —C1-C8 alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl, —C3-C12 cycloalkyl, substituted —C3-C12 cycloalkyl, —C3-C12 cycloalkenyl, or substituted —C3-C12 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, the chemical Examples provided hereinbelow provide synthetic schemes for the preparation of the compound JQ1 (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 substitution of appropriate starting materials.
For example, starting from JQ1, the analogous amine can be prepared as shown in Scheme 1, below.




As shown in Scheme 1, hydrolysis of the t-butyl ester of JQ1 affords the carboxylic acid, which is treated with diphenylphosphoryl azide (DPPA) and subjected to Curtius rearrangement 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 yields secondary amines, which can be further alkylated to provide tertiary amines.




Scheme 2 shows the synthesis of further examples of the compounds of the invention, e.g., of Formula I, in which the fused ring core is modified (e.g., by substitution of a different aromatic ring as Ring A in Formula I). Use of aminodiarylketones having appropriate functionality (e.g., in place of the aminodiarylketone S2 in Scheme 51, infra) provides new compounds having a variety of fused ring cores and/or aryl group appendages (corresponding to group R in Formula I). Such aminodiarylketones are commercially available or can be prepared by a variety of methods, some of which are known in the art.
Scheme 3 provides additional exemplary synthetic schemes for preparing further compounds of the invention.




As shown in Scheme 3, a fused bicyclic precursor (see Scheme 51, infra, for synthesis of this compound) is functionalized with a moiety R (DAM=dimethylaminomethylene protecting group) and then elaborated by reaction with a hydrazide to form the tricyclic fused core. Substituent Rx can be varied by selection of a suitable hydrazide.
Additional examples of compounds of the invention (which can be prepared by the methods described herein) include:
Amides:
Amides can be prepared, e.g., by preparation of a corresponding carboxylic acid or ester, followed by amidation with an appropriate amine using standard conditions. In certain embodiments, an amide provides a two-carbon “linker” with a terminal terminal nitrogen-containing ring (e.g., pyridyl, piperidyl, piperazinyl, imidazolyl (including N-methyl-imidazolyl), morpholinyl, and the like. Exemplary amide structures include:







The use of a two-carbon linker between the amide moiety and the terminal nitrogen-containing ring is preferred.
“Reverse Amides”:







Secondary Amines:







Boronic Acids:




In certain embodiments, a compound having at least one chiral center is present in racemic form. In certain embodiments, a compound having at least one chiral center is enantiomerically 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 configuration as the compound (+)-JQ1 ((S)-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) described herein. In certain embodiments of any of the Formulae disclosed herein, the compound is not represented by the following structure:




in which:
R′1, is C1-C4 alkyl;
R′2 is hydrogen, halogen, or C1-C4 alkyl optionally substituted with a halogen atom or a hydroxyl group;
R′3 is a halogen atom, phenyl optionally substituted by a halogen atom, C1-C4 alkyl, C1-C4 alkoxyy, or cyano; —NR5—(CH2)m—R6 wherein R5 is a hydrogen atom or C1-C4 alkyl, m is an integer of 0-4, and R6 is phenyl or pyridyl optionally substituted by a halogen atom; or —NR7—CO—(CH2)n—R8 wherein R7 is a hydrogen atom or C1-C4 alkyl, n is an integer of 0-2, and R8 is phenyl or pyridyl optionally substituted by a halogen atom; and
R′4 is —(CH2)a—CO—NH—R9 wherein a is an integer of 1-4, and R9 is C1-C4 alkyl; C1-C4 hydroxyalkyl; C1-C4 alkoxy; or phenyl or pyridyl optionally substituted by C1-C4 alkyl, C1-C4 alkoxy, amino or a hydroxyl group or —(CH2)b—COOR10 wherein b is an integer of 1-4, and R10 is C1-C4 alkyl.
The term “pharmaceutically acceptable salt” also refers to a salt prepared from a compound disclosed herein (e.g., JQ1, 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 acceptable inorganic or organic base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine; tributyl amine; pyridine; N-methyl,N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkyl amines), such as mono-, bis-, or tris-(2-hydroxyethyl)-amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N,-di-lower alkyl-N-(hydroxy lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)-amine, or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like. The term “pharmaceutically acceptable salt” also refers to a salt prepared from a compound disclosed herein, or any other compound delineated herein, having a basic functional group, such as an amino functional group, and a pharmaceutically acceptable inorganic or organic acid. Suitable acids include, but are not limited to, hydrogen sulfate, citric acid, acetic acid, oxalic acid, hydrochloric acid, hydrogen bromide, hydrogen iodide, nitric acid, phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbic acid, succinic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucaronic acid, saccharic acid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.
Methods of the Invention
The present invention also relates to using the novel compounds described herein, as well as other inhibitors of BRDT as male contraceptives. Such compounds are known in the art and described, for example, in WO2009084693 or corresponding US2010286127.
Thus, in one aspect, the invention provides methods for reducing or inhibiting spermatozoa emission involving administering an effective amount of a BRDT inhibitor to a male subject. In embodiments, the inhibitor is a compound having a formula delineated herein, a derivative thereof, or a pharmaceutically acceptable salt or prodrug thereof.
In embodiments, the methods involve administering the inhibitor in an amount sufficient to suppress spermatogenesis.
In embodiments, the methods involve administering the inhibitor in an amount sufficient to induce azoospermia or oligozoospermia.
In embodiments, the methods involve administering the inhibitor in an amount sufficient to lower the spermatozoa concentration to not more than 3 million/mL, 2 million/mL, 1 million/mL, 0.5 million/mL, 0.25 million/mL, or 0.1 million/mL. In related embodiments, the methods involve administering the inhibitor in an amount sufficient to lower the spermatozoa concentration to not more than 0.1 million/mL.
In embodiments, the inhibitor is adminstered in combination with a pharmaceutically acceptable carrier, excipient, or diluent.
In embodiments, the inhibitor is administered to the subject orally, transdermally, or by injection. In related embodiments, the inhibitor is administered in the form of a tablet or capsule. In related embodiments, the inhibitor is administered by parenteral injection, intramuscular injection, intravenous injection, subcutaneous implantation, subcutaneous injection, or transdermal preparation.
In embodiments, the inhibitor is used in combination with at least one additional male contraceptive agent or device. In related embodiments, the additional male contraceptive is a condom. In other related embodiments, the additional male contraceptive is a modulator of testosterone production, androgen receptor function or stability.
Pharmaceutical Compositions
The invention features pharmaceutical compositions that contain one or more of the compounds described herein, a derivative thereof, or a pharmaceutically acceptable salt or prodrug thereof as the active ingredient(s). The pharmaceutical compositions contain a pharmaceutically acceptable carrier, excipient, or diluent, which includes any pharmaceutical agent that does not itself induce the production of an immune response harmful to a subject receiving the composition, and which may be administered without undue toxicity. As used herein, the term “pharmaceutically acceptable” means being approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopia, European Pharmacopia or other generally recognized pharmacopia for use in mammals, and more particularly in humans. These compositions can be useful as a male contraceptive.
A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in Remington's Pharmaceutical Sciences (17th ed., Mack Publishing Company) and Remington: The Science and Practice of Pharmacy (21st ed., Lippincott Williams & Wilkins), which are hereby incorporated by reference. The formulation of the pharmaceutical composition should suit the mode of administration. In embodiments, the pharmaceutical composition is suitable for administration to humans, and can be sterile, non-particulate and/or non-pyrogenic.
Pharmaceutically acceptable carriers, excipients, or diluents include, but are not limited, to saline, buffered saline, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof.
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, and antioxidants can also be present in the compositions.
Examples of pharmaceutically-acceptable antioxidants include, but are not limited to: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
In embodiments, the pharmaceutical composition is provided in a solid form, such as a lyophilized powder suitable for reconstitution, a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
In embodiments, the pharmaceutical composition is supplied in liquid form, for example, in a sealed container indicating the quantity and concentration of the active ingredient in the pharmaceutical composition. In related embodiments, the liquid form of the pharmaceutical composition is supplied in a hermetically sealed container.
Methods for formulating the pharmaceutical compositions of the present invention are conventional and well-known in the art (see Remington and Remington's). One of skill in the art can readily formulate a pharmaceutical composition having the desired characteristics (e.g., route of administration, biosafety, and release profile).
Methods for preparing the pharmaceutical compositions include the step of bringing into association the active ingredient with a pharmaceutically acceptable carrier and, optionally, one or more accessory ingredients. The pharmaceutical compositions can be prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. Additional methodology for preparing the pharmaceutical compositions, including the preparation of multilayer dosage forms, are described in Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (9th ed., Lippincott Williams & Wilkins), which is hereby incorporated by reference.
Methods of Delivery
The pharmaceutical compositions of the present invention can be administered to a subject by oral and non-oral means (e.g., topically, transdermally, or by injection). Such modes of administration and the methods for preparing an appropriate pharmaceutical composition for use therein are described in Gibaldi's Drug Delivery Systems in Pharmaceutical Care (1st ed., American Society of Health-System Pharmacists), which is hereby incorporated by reference.
In embodiments, the pharmaceutical compositions are administered orally in a solid form.
Pharmaceutical compositions suitable for oral administration can be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound(s) described herein, a derivative thereof, or a pharmaceutically acceptable salt or prodrug thereof as the active ingredient(s). The active ingredient can also be administered as a bolus, electuary, or paste.
In solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, excipients, or diluents, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions can also comprise buffering agents. Solid compositions of a similar type can also be prepared using fillers in soft and hard-filled gelatin capsules, and excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (for example, gelatin or hydroxypropylmethyl cellulose), lubricants, inert diluents, preservatives, disintegrants (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-actives, and/or dispersing agents. Molded tablets can be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
The tablets and other solid dosage forms, such as dragees, capsules, pills, and granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well-known in the art.
The pharmaceutical compositions can also be formulated so as to provide slow, extended, or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. The pharmaceutical compositions can also optionally contain opacifying agents and may be of a composition that releases the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more pharmaceutically acceptable carriers, excipients, or diluents well-known in the art (see, e.g., Remington and Remington's).
The pharmaceutical compositions can be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
In embodiments, the pharmaceutical compositions are administered orally in a liquid form.
Liquid dosage forms for oral administration of an active ingredient include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms can contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the liquid pharmaceutical compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents, and the like.
Suspensions, in addition to the active ingredient(s) can contain suspending agents such as, but not limited to, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
In embodiments, the pharmaceutical compositions are administered by non-oral means such as by topical application, transdermal application, injection, and the like. In related embodiments, the pharmaceutical compositions are administered parenterally by injection, infusion, or implantation (e.g., intravenous, intramuscular, intraarticular, subcutaneous, and the like).
Compositions for parenteral use can be presented in unit dosage forms, e.g. in ampoules or in vials containing several doses, and in which a suitable preservative can be added. Such compositions can be in form of a solution, a suspension, an emulsion, an infusion device, a delivery device for implantation, or it can be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. One or more co-vehicles, such as ethanol, can also be employed. Apart from the active ingredient(s), the compositions can contain suitable parenterally acceptable carriers and/or excipients or the active ingredient(s) can be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the compositions can also contain suspending, solubilising, stabilising, pH-adjusting agents, and/or dispersing agents.
The pharmaceutical compositions can be in the form of sterile injections. To prepare such a composition, the active ingredient is dissolved or suspended in a parenterally acceptable liquid vehicle. Exemplary vehicles and solvents include, but are not limited to, water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution and isotonic sodium chloride solution. The pharmaceutical composition can also contain one or more preservatives, for example, methyl, ethyl or n-propyl p-hydroxybenzoate. To improve solubility, a dissolution enhancing or solubilising agent can be added or the solvent can contain 10-60% w/w of propylene glycol or the like.
The pharmaceutical compositions can contain one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders, which can be reconstituted into sterile injectable solutions or dispersions just prior to use. Such pharmaceutical compositions can contain antioxidants; buffers; bacteriostats; solutes, which render the formulation isotonic with the blood of the intended recipient; suspending agents; thickening agents; preservatives; and the like.
Examples of suitable aqueous and nonaqueous carriers, which can be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
In some embodiments, in order to prolong the effect of an active ingredient, it is desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the active ingredient then depends upon its rate of dissolution which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered active ingredient is accomplished by dissolving or suspending the compound in an oil vehicle. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
Controlled release parenteral compositions can be in form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, emulsions, or the active ingredient can be incorporated in biocompatible carrier(s), liposomes, nanoparticles, implants or infusion devices.
Materials for use in the preparation of microspheres and/or microcapsules include biodegradable/bioerodible polymers such as polyglactin, poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutamine) and poly(lactic acid).
Biocompatible carriers which can be used when formulating a controlled release parenteral formulation include carbohydrates such as dextrans, proteins such as albumin, lipoproteins or antibodies.
Materials for use in implants can be non-biodegradable, e.g., polydimethylsiloxane, or biodegradable such as, e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters).
In embodiments, the active ingredient(s) are administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation, or solid particles containing the compound. A nonaqueous (e.g., fluorocarbon propellant) suspension can be used. The pharmaceutical composition can also be administered using a sonic nebulizer, which would minimize exposing the agent to shear, which can result in degradation of the compound.
Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the active ingredient(s) together with conventional pharmaceutically-acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.
Dosage forms for topical or transdermal administration of an active ingredient(s) includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active ingredient(s) can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants as appropriate.
Transdermal patches suitable for use in the present invention are disclosed in Transdermal Drug Delivery: Developmental Issues and Research Initiatives (Marcel Dekker Inc., 1989) and U.S. Pat. Nos. 4,743,249, 4,906,169, 5,198,223, 4,816,540, 5,422,119, 5,023,084, which are hereby incorporated by reference. The transdermal patch can also be any transdermal patch well-known in the art, including transscrotal patches. Pharmaceutical compositions in such transdermal patches can contain one or more absorption enhancers or skin permeation enhancers well-known in the art (see, e.g., U.S. Pat. Nos. 4,379,454 and 4,973,468, which are hereby incorporated by reference). Transdermal therapeutic systems for use in the present invention can be based on iontophoresis, diffusion, or a combination of these two effects.
Transdermal patches have the added advantage of providing controlled delivery of active ingredient(s) to the body. Such dosage forms can be made by dissolving or dispersing the active ingredient(s) in a proper medium. Absorption enhancers can also be used to increase the flux of the active ingredient across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active ingredient(s) in a polymer matrix or gel.
Such pharmaceutical compositions can be in the form of creams, ointments, lotions, liniments, gels, hydrogels, solutions, suspensions, sticks, sprays, pastes, plasters and other kinds of transdermal drug delivery systems. The compositions can also include pharmaceutically acceptable carriers or excipients such as emulsifying agents, antioxidants, buffering agents, preservatives, humectants, penetration enhancers, chelating agents, gel-forming agents, ointment bases, perfumes, and skin protective agents.
Examples of emulsifying agents include, but are not limited to, naturally occurring gums, e.g. gum acacia or gum tragacanth, naturally occurring phosphatides, e.g. soybean lecithin and sorbitan monooleate derivatives.
Examples of antioxidants include, but are not limited to, butylated hydroxy anisole (BHA), ascorbic acid and derivatives thereof, tocopherol and derivatives thereof, and cysteine.
Examples of preservatives include, but are not limited to, parabens, such as methyl or propyl p-hydroxybenzoate and benzalkonium chloride.
Examples of humectants include, but are not limited to, glycerin, propylene glycol, sorbitol and urea.
Examples of penetration enhancers include, but are not limited to, propylene glycol, DMSO, triethanolamine, N,N-dimethylacetamide, N,N-dimethylformamide, 2-pyrrolidone and derivatives thereof, tetrahydrofurfuryl alcohol, propylene glycol, diethylene glycol monoethyl or monomethyl ether with propylene glycol monolaurate or methyl laurate, eucalyptol, lecithin, Transcutol®, and Azone®.
Examples of chelating agents include, but are not limited to, sodium EDTA, citric acid and phosphoric acid.
Examples of gel forming agents include, but are not limited to, Carbopol, cellulose derivatives, bentonite, alginates, gelatin and polyvinylpyrrolidone.
In addition to the active ingredient(s), the ointments, pastes, creams, and gels of the present invention can contain excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons, such as butane and propane.
Injectable depot forms are made by forming microencapsule matrices of compound(s) of the invention in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of compound to polymer, and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
Subcutaneous implants are well-known in the art and are suitable for use in the present invention. Subcutaneous implantation methods are preferably non-irritating and mechanically resilient. The implants can be of matrix type, of reservoir type, or hybrids thereof. In matrix type devices, the carrier material can be porous or non-porous, solid or semi-solid, and permeable or impermeable to the active compound or compounds. The carrier material can be biodegradable or may slowly erode after administration. In some instances, the matrix is non-degradable but instead relies on the diffusion of the active compound through the matrix for the carrier material to degrade. Alternative subcutaneous implant methods utilize reservoir devices where the active compound or compounds are surrounded by a rate controlling membrane, e.g., a membrane independent of component concentration (possessing zero-order kinetics). Devices consisting of a matrix surrounded by a rate controlling membrane also suitable for use.
Both reservoir and matrix type devices can contain materials such as polydimethylsiloxane, such as SilasticTM, or other silicone rubbers. Matrix materials can be insoluble polypropylene, polyethylene, polyvinyl chloride, ethylvinyl acetate, polystyrene and polymethacrylate, as well as glycerol esters of the glycerol palmitostearate, glycerol stearate, and glycerol behenate type. Materials can be hydrophobic or hydrophilic polymers and optionally contain solubilising agents.
Subcutaneous implant devices can be slow-release capsules made with any suitable polymer, e.g., as described in U.S. Pat. Nos. 5,035,891 and 4,210,644, which are hereby incorporated by reference.
In general, at least four different approaches are applicable in order to provide rate control over the release and transdermal permeation of a drug compound. These approaches are: membrane-moderated systems, adhesive diffusion-controlled systems, matrix dispersion-type systems and microreservoir systems. It is appreciated that a controlled release percutaneous and/or topical composition can be obtained by using a suitable mixture of these approaches.
In a membrane-moderated system, the active ingredient is present in a reservoir which is totally encapsulated in a shallow compartment molded from a drug-impermeable laminate, such as a metallic plastic laminate, and a rate-controlling polymeric membrane such as a microporous or a non-porous polymeric membrane, e.g., ethylene-vinyl acetate copolymer. The active ingredient is released through the ratecontrolling polymeric membrane. In the drug reservoir, the active ingredient can either be dispersed in a solid polymer matrix or suspended in an unleachable, viscous liquid medium such as silicone fluid. On the external surface of the polymeric membrane, a thin layer of an adhesive polymer is applied to achieve an intimate contact of the transdermal system with the skin surface. The adhesive polymer is preferably a polymer which is hypoallergenic and compatible with the active drug substance.
In an adhesive diffusion-controlled system, a reservoir of the active ingredient is formed by directly dispersing the active ingredient in an adhesive polymer and then by, e.g., solvent casting, spreading the adhesive containing the active ingredient ance onto a flat sheet of substantially drug-impermeable metallic plastic backing to form a thin drug reservoir layer.
A matrix dispersion-type system is characterized in that a reservoir of the active ingredient is formed by substantially homogeneously dispersing the active ingredient in a hydrophilic or lipophilic polymer matrix. The drug-containing polymer is then molded into disc with a substantially well-defined surface area and controlled thickness. The adhesive polymer is spread along the circumference to form a strip of adhesive around the disc.
A microreservoir system can be considered as a combination of the reservoir and matrix dispersion type systems. In this case, the reservoir of the active substance is formed by first suspending the drug solids in an aqueous solution of water-soluble polymer and then dispersing the drug suspension in a lipophilic polymer to form a multiplicity of unleachable, microscopic spheres of drug reservoirs.
Any of the above-described controlled release, extended release, and sustained release compositions can be formulated to release the active ingredient in about 30 minutes to about 1 week, in about 30 minutes to about 72 hours, in about 30 minutes to 24 hours, in about 30 minutes to 12 hours, in about 30 minutes to 6 hours, in about 30 minutes to 4 hours, and in about 3 hours to 10 hours. In embodiments, an effective concentration of the active ingredient(s) is sustained in a subject for 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, or more after administration of the pharmaceutical compositions to the subject.
Methods of Delivery
When the compound(s) of the invention are administered as pharmaceuticals to humans and animals, they can be given per se or as a pharmaceutical composition containing active ingredient in combination with a pharmaceutically acceptable carrier, excipient, or diluent.
Actual dosage levels and time course of administration of the active ingredients in the pharmaceutical compositions of the invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. Generally, compounds or pharmaceutical compositions of the invention are administered in an effective amount or quantity sufficient to reduce or inhibit spermatozoa emission in a male subject. In embodiments, administration of the compound or pharmaceutical composition suppresses spermatogenesis, induces azoospermia, or induces oligozoospermia.
Exemplary dose ranges include 0.01 mg to 250 mg per day, 0.01 mg to 100 mg per day, 1 mg to 100 mg per day, 10 mg to 100 mg per day, 1 mg to 10 mg per day, and 0.01 mg to 10 mg per day. A preferred dose of the compound of the invention is the maximum that a patient can tolerate and not develop serious or unacceptable side effects. In embodiments, the compound(s) of the present invention is administered at a concentration of about 10 micrograms to about 100 mg per kilogram of body weight per day, about 0.1 to about 10 mg/kg per day, or about 1.0 mg to about 10 mg/kg of body weight per day. In embodiments, the pharmaceutical composition comprises a compound(s) of the invention in an amount ranging between 1 and 10 mg, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg.
In embodiments, the therapeutically effective dosage produces a serum concentration of compound of from about 0.1 ng/ml to about 50-100 μg/ml. The pharmaceutical compositions typically should provide a dosage of from about 0.001 mg to about 2000 mg of compound per kilogram of body weight per day. For example, dosages for systemic administration to a human patient can range from 1-10 μg/kg, 20-80 μg/kg, 5-50 μg/kg, 75-150 μg/kg, 100-500 μg/kg, 250-750 μg/kg, 500-1000 μg/kg, 1-10 mg/kg, 5-50 mg/kg, 25-75 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 50-100 mg/kg, 250-500 mg/kg, 500-750 mg/kg, 750-1000 mg/kg, 1000-1500 mg/kg, 1500-2000 mg/kg, 5 mg/kg, 20 mg/kg, 50 mg/kg, 100 mg/kg, 500 mg/kg, 1000 mg/kg, 1500 mg/kg, or 2000 mg/kg. Pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 5000 mg, for example from about 100 to about 2500 mg of the compound or a combination of essential ingredients per dosage unit form.
In embodiments, the pharmaceutical composition comprises a compound(s) of the invention in an amount sufficient to lower spermatozoa concentration to not more than 3 million/mL of semen, such as not more than 2 million/mL, 1 million/mL, 0.5 million/mL, 0.25 million/mL, or 0.1 million/mL. In related embodiments, the pharmaceutical composition comprises a compound(s) of the invention in an amount sufficient to lower spermatozoa concentration to not more than 0.1 million/mL.
Determination of an effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, an efficacious or effective amount of a compound(s) of the invention is determined by first administering a low dose of the compound(s) and then incrementally increasing the administered dose or dosages until a desired effect (e.g., decreased spermatozoa levels in seminal fluid) is observed in the treated subject, with minimal or acceptable toxic side effects. Applicable methods for determining an appropriate dose and dosing schedule for administration of a pharmaceutical composition of the present invention are described, for example, in Goodman and Gilman's The Pharmacological Basis of Therapeutics, Goodman et al., eds., 11th Edition, McGraw-Hill 2005, and Remington: The Science and Practice of Pharmacy, 20th and 21st Editions, Gennaro and University of the Sciences in Philadelphia, Eds., Lippencott Williams & Wilkins (2003 and 2005), which are hereby incorporated by reference.
Kits
The invention provides for a kit for effecting male contraception. In embodiments, the kit contains one or more of the compounds or pharmaceutical compositions described herein. In embodiments, the kit provides instructions for use. The instructions for use can pertain to any of the methods described herein. In related embodiments, the instructions pertain to using the compound(s) or pharmaceutical composition(s) for reducing or inhibiting spermatozoa emission. In embodiments, the kit provides a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale of the kit and the components therein for human administration.
The invention also provides for compound(s) or pharmaceutical composition(s) packaged in a hermetically sealed container (e.g., ampoule or sachette) indicating the quantity of compound. In embodiments, a compound or pharmaceutical composition is supplied as a liquid. In other embodiments, a compound or pharmaceutical composition is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline, to the appropriate concentration for administration to a subject.
The invention also provides for transdermal patches containing the compound(s) or pharmaceutical composition(s).
In embodiments, the kit provides compound(s) or pharmaceutical composition(s) in more than one dosage unit. The kit can contain from 1 to about 120 or more, from 1 to about 60, from 1 to about 30, from 1 to about 10, or from 1 to about 7 dosage units. In cases where the compound(s) or pharmaceutical composition(s) is adapted to release a therapeutically effective amount of the active ingredient over a 24 hour period, the kit conveniently comprises 1, about 5, about 7, about 10, about 14, or about 30 dosage units. In cases where the compound(s) or pharmaceutical composition(s) is adapted to provide a therapeutically effective amount of the active ingredient over a 12 hour period, the kit conveniently comprises 1, 2, about 10, about 14, about 30 or about 60 dosage units. In cases where the compound(s) or pharmaceutical composition(s) is adapted to provide a therapeutically effective amount of the active ingredient over an about 3 to about 10 hour (e.g., about a 6 or 8 hour) period, the kit comprises about 1, about 4, about 40, about 60 or about 120 dosage units. One skilled in the art will recognize that other numbers of dosage units can be included in the kit without departing materially from the present invention.
Screening Methods
As described herein, the invention provides specific examples of chemical compounds, including JQ1, as well as other substituted compounds that bind a bromodomain binding pocket and are useful as a male contraceptive. However, the invention is not so limited. The invention further provides a simple means for identifying agents (including nucleic acids, peptides, small molecule inhibitors, and mimetics) that are capable of inhibiting spermatogenesis. Such compounds are also expected to be useful as male contraceptives.
In particular embodiments, the effect of a compound or other agent of the invention is analyzed by assaying spermatogenesis. Agents and compounds of the invention that reduce spermatogenesis are identified as useful as male contraceptives.
Virtually any agent that specifically binds to a BET family member or that reduces the biological activity of a BET family member may be employed in the methods of the invention. Methods of the invention are useful for the high-throughput low-cost screening of candidate agents that reduce or otherwise inhibit spermatogenesis. A candidate agent that specifically binds to a bromodomain of a BET family member is then isolated and tested for activity in an in vitro assay or in vivo assay for its ability to inhibit spermatogenesis. One skilled in the art appreciates that the effects of a candidate agent on a cell is typically compared to a corresponding control cell not contacted with the candidate agent. Thus, the screening methods include comparing spermatogenesis in a testes contacted by a candidate agent to the spermatogenesis present in an untreated control testes.
Once identified, agents of the invention (e.g., agents that specifically bind to and/or antagonize a bromodomain) may be used as male contraceptives. Potential bromodomain antagonists include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acid ligands, aptamers, and antibodies that bind to a BET family member bromodomain and reduce its activity. Candidate agents may be tested for their ability to reduce spermatogenesis.
Test Compounds and Extracts
In certain embodiments, BET family member antagonists (e.g., agents that specifically bind and reduce the activity of a bromodomain) are identified from large libraries of natural product or synthetic (or semi-synthetic) extracts or chemical libraries or from polypeptide or nucleic acid libraries, according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Viirtually any number of unknown chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as the modification of existing polypeptides.
Libraries of natural polypeptides in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Such polypeptides can be modified to include a protein transduction domain using methods known in the art and described herein. In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Examples of methods for the synthesis of molecular 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. Med. Chem. 37:2678, 1994; Cho et al., Science 261:1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl. 33:2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061, 1994; and Gallop et al., J. Med. Chem. 37:1233, 1994. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.
Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of polypeptides, chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, chemical compounds to be used as candidate compounds can be synthesized from readily available starting materials using standard synthetic techniques and methodologies known to those of ordinary skill in the art. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds identified by the methods described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.
W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.
Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA 89:1865-1869, 1992) or on phage (Scott and Smith, Science 249:386-390, 1990; Devlin, Science 249:404-406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. 87:6378-6382, 1990; Felici, J. Mol. Biol. 222:301-310, 1991; Ladner supra.).
In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their activity should be employed whenever possible.
When a crude extract is found to have BET family member bromodomain binding activity further fractionation of the positive lead extract is necessary to isolate molecular constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract that reduces spermatogenesis. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful as therapeutics are chemically modified according to methods known in the art.
EXAMPLES
It should be appreciated that the invention should not be construed to be limited to the examples that are now described; rather, the invention should be construed to include any and all applications provided herein and all equivalent variations within the skill of the ordinary artisan.
I. Chemical Examples
Synthesis and Methods of Preparation
Compounds of the invention can be synthesized by methods described herein, and/or according to methods known to one of ordinary skill in the art in view of the description herein.







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 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 (20 ml, 0.35 M) at 23° C.21. The mixture was then heated to 70° C. After 12 hours, the reaction mixture was cooled to 23° C. and poured into brine (100 ml). The aqueous layer was extracted with ethyl acetate (3×50 ml). The combined organic layers were washed with brine (50 ml), were dried over anhydrous sodium sulphate, were filtered, and were concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 40 gram silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford S2 (1.28 g, 70%) as a yellow solid.
(S)-tert-Butyl-3-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-4-{[3-(4-chlorobenzoyl)-4,5-dimethylthiophen-2-yl]amino}-4-oxobutanoate (S3)
(2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) (827 mg, 2.0 mmol, 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 β-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 then stirred at 23° C. for 5 min. S2 (266 mg, 1.0 mmol, 1 equiv) was then added as a solid. The reaction mixture was stirred at 23° C. After 16 hours, ethyl acetate (20 ml) and brine (20 ml) were added. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2×20 ml). The combined organic layers were washed with brine (30 ml), were dried over with anhydrous sodium sulphate, were filtered, and were concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF, 40 gram silica gel, gradient 0 to 100% ethyl 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)
Compound S3 (560 mg, 0.85 mmol, 1 equiv) was dissolved into 20% piperidine in DMF solution (4.0 ml, 0.22 M) at 23° C. After 30 min, ethyl acetate (20 ml) and brine (20 ml) were added to the reaction mixture. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2×20 ml). The combined organic layers were washed with brine (3×25 ml), were dried over anhydrous sodium sulphate, were filtered, and were concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 24 gram silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford free amine S4 (370 mg, 90%) as yellow solid. The enantiomeric purity dropped to 75% (determined with Berger Supercritical Fluid Chromatography (SFC) using AS-H column).
(S)-tert-Butyl 2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetate (S5)
Amino ketone (S4) (280 mg, 0.63 mmol) was dissolved in 10% acetic acid ethanol solution (21 ml, 0.03 M). The reaction mixture was heated to 85° C. After 30 minutes, all solvents were removed under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 12 gram silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford compound S5 (241 mg, 95%) as white solid. Enantiomeric purity of S5 was 67% (determined with Berger Supercritical Fluid Chromatography (SFC) using an AS-H column).
tert-Butyl 2-(5-(4-chlorophenyl)-6,7-dimethyl-2-thioxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetate (S6)
Phosphorus pentasulfide (222 mg, 1.0 mmol, 2.00 equiv), 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 16 h, brine (20 ml) and ethyl acetate (35 ml) were added. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (3×30 ml). The combined organic layers were washed with brine (2×15 ml), were dried over anhydrous sodium sulphate, were filtered, and were concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 24 gram silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford S6 (141 mg, 65%) as brown solid with recovered S5 (73 mg, 34%).
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 a solution of S6 (158 mg, 0.36 mmol, 1 equiv) in THF (2.6 ml, 0.14 M) at 0° C. The reaction mixture was warmed to 23° C., and stirred at 23° C. for 1 h. All solvents were removed under reduced pressure. The resulting hydrazine was used directly without purification. The hydrazine was then dissolved in a 2:3 mixture of trimethyl orthoacetate and toluene (6 ml, 0.06 M). The reaction mixture was heated to 120° C. After 2 h, all the solvents were removed under reduced pressure. The residue was purified by flash column chromatography (Combiflash system, 4 g silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford JQ1 (140 mg, 85% in 2 steps) as white solid. The reaction conditions further epimerized the stereogenic center, resulting in the racemate, JQ1 (determined with Berger Supercritical Fluid Chromatography (SFC) with an AS-H column).




(Benzotriazol-1-yloxyl)tripyrrolidinophosphonium (PyBOP) (494 mg, 0.95 mmol, 0.95 equiv), N,N-diisopropylethylamine (0.50 ml, 2.8 mmol, 2.75 equiv) were added sequentially to a solution of 9-fluorenylmethoxycarbonyl-aspartic acid13-tert-butyl ester[Fmoc-Asp(Ot-Bu)-OH] (411 mg, 1.00 mmol, 1.0 equiv) in N,N-dimethylformamide (1.0 ml, 1.0 M). The mixture was then stirred at 23° C. for 5 min. S2 (266 mg, 1.0 mmol, 1 equiv) was then added as solid. The reaction mixture was stirred at 23° C. After 4 h, ethyl acetate (20 ml) and brine (20 ml) were added. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2×20 ml). The combined organic layers were washed with brine, were dried over with anhydrous sodium sulphate, were filtered, and were concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 40 gram silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford S3 (452 mg, 72%) as brown oil.
(S)-tert-butyl 3-amino-4-((3-(4-chlorobenzoyl)-4,5-dimethylthiophen-2-yl)amino)-4-oxobutanoate (S4)
Compound S3 (310 mg, 0.47 mmol, 1 equiv) was dissolved into 20% piperidine in DMF solution (2.2 ml, 0.22 M) at 23° C. After 30 min, ethyl acetate (20 ml) and brine (20 ml) were added to the reaction mixture. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2×20 ml). The combined organic layers were washed with brine (3×25 ml), were dried over anhydrous sodium sulphate, were filtered, and were concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 24 gram silica gel, gradient 0 to 100% ethyl acetate-hexane) to afford free amine S4 (184 mg, 90%) 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-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, 0.04 M). Silica gel (300 mg) was added, and 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 were concentrated. The residue was purified by flash column chromatography (Combiflash RF system, 12 gram silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford compound 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-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate[(+)JQ1]
Potassium tert-butoxide (1.0 M solution in THF, 0.3 ml, 0.30 mmol, 1.10 equiv) was added to a solution of S5 (114 mg, 0.27 mmol, 1 equiv) in THF (1.8 ml, 0.15 M) at −78° C. The reaction mixture was warmed to −10° C., and stirred at 23° C. for 30 min. The reaction mixture was cooled to −78° C. Diethyl chlorophosphate (0.047 ml, 0.32 mmol, 1.20 equiv) was added to reaction mixture22. The resulting mixture was 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 90° C. After 1 h, all solvents were removed under reduce pressure. The residue was purified with flash column chromatography (Combiflash system, 4 g silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford (+)-JQ1 (114 mg, 92%) as white solid with 90% enantiomeric purity (determined with Berger Supercritical Fluid Chromatography (SFC) using AS-H column, 85% hexanes-methanol, 210 nm, tR (R-enantiomer)=1.59 min, tR (S-enantiomer)=3.67 min). The product was further purified by chiral preparative HPLC (Agilent High Pressure Liquid Chromatography using an OD-H column) to provide the S-enantiomer in greater than 99% ee.
1H NMR (600 MHz, CDCl3, 25° C.) δ 7.39 (d, J=8.4 Hz, 2H), 7.31 (d, J=8.4 Hz, 2H), 4.54 (t, J=6.6 MHz, 1H), 3.54-3.52 (m, 2H), 2.66 (s, 3H), 2.39 (s, 3H), 1.67 (s, 3H), 1.48 (s, 9H).
13C NMR (150 MHz, CDCl3, 25° C.) δ 171.0, 163.8, 155.7, 150.0, 136.9, 131.1, 130.9, 130.6, 130.3, 128.9, 81.2, 54.1, 38.1, 28.4, 14.6, 13.5, 12.1.
HRMS (ESI) calc'd for C21H24ClN2O3S [M+H]+: 457.1460, found 457.1451 m/z.
TLC (EtOAc), Rf: 0.32 (UV)
[α]22D (c (0.5, CHCl3)
(−)-JQ1 was synthesized in a similar manner, employing Fmoc-D-Asp(Ot-Bu)-OH as a starting material, and was further purified by chiral preparative HPLC (Agilent High Pressure Liquid Chromatography using an OD-H column) to afford the R-enantiomer in greater than 99% ee. [α]22D=−72 (c 0.5, CHCl3)
Synthesis of Additional Compounds
Additional compounds of the invention were prepared as illustrated in Scheme S3.




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







Additional compounds were prepared as shown in the table below:












Compound

MS [M + H]+


Name
Structure
m/z (Observed)

















(S)-JQ1




457.1


 


(R)-JQ1




457.1


 


JQ3




415.1


 


JQ4




519.1


 


JQ6




493.1


 


JQ7




579.0


 


JQ8




494.1


 


JQ10




501.1


 


JQ11




511.1


 


JQ1-FITC




804.1


 


JQ1-Biotin




829.3


 


JQ13




526.2


 


KS1




429.1


 


JQ18



   Chemical Formula: C24H27ClN4O3S Exact Mass: 486.14924 Molecular Weight: 487.01418
487.1


 


JQ19



   Chemical Formula: C24H27ClN4O2S Exact Mass: 470.15432 Molecular Weight: 471.01478
471.1


 


JQ20



   JQI-II-023 Chemical Formula: C19H19ClN4S Exact Mass: 370.10190 Molecular Weight: 370.89896
370.1


 


JQ21



   JQI-II-024 Chemical Formula: C22H23ClN4O2S Exact Mass: 442.12302 Molecular Weight: 442.96162
443.1


 


JQ24A



   Chemical Formula: C24H26ClN3O2S Exact Mass: 455.1434 Molecular Weight: 456.0001
456.1


 


JQ24B



   Chemical Formula: C24H26ClN3O2S Exact Mass: 455.1434 Molecular Weight: 456.0001
456.1


 


JQ25



   Chemical Formula: C26H24ClN5O2S Exact Mass: 505.1339 Molecular Weight: 506.0191
506.1


 


JQB



   Chemical Formula: C23H24N4O2 Exact Mass: 388.1899 Molecular Weight: 388.4623
389.2


 


JQ30



   Chemical Formula: C23H26ClN5OS Exact Mass: 455.1547 Molecular Weight: 456.0034
456.2


 


JQ31



   Chemical Formula: C23H26ClN5OS Exact Mass: 455.1547 Molecular Weight: 456.0034
456.2


 


JQ32



   Chemical Formula: C20H17ClF3N5OS Exact Mass: 467.0794 Molecular Weight: 467.8951
468.1


 


JQ33



   Chemical Formula: C25H29ClN6O2S Exact Mass: 512.1761 Molecular Weight: 513.0548
512.2


 


JQ34



   Chemical Formula: C26H25ClN6OS Exact Mass: 504.1499 Molecular Weight: 505.0343
505.1


 


JQ35



   Chemical Formula: C27H34ClN7OS Exact Mass: 539.2234 Molecular Weight: 540.1232
540.2


 


JQ36



   Chemical Formula: C27H34ClN7OS Exact Mass: 539.2234 Molecular Weight: 540.1232
540.2


 


JQ37



   Chemical Formula: C22H25N5O2S Exact Mass: 423.1729 Molecular Weight: 423.5312
424.2


 


JQ38



   Chemical Formula: C25H26ClN7OS Exact Mass: 507.1608 Molecular Weight: 508.0382
508.2


 


JQ39



   Chemical Formula: C26H25ClN6OS Exact Mass: 504.1499 Molecular Weight: 505.0343
505.1


 


JQ40



   Chemical Formula: C25H30ClN7OS Exact Mass: 511.1921 Molecular Weight: 512.0700
512.2


 


JQ41



   Chemical Formula: C27H34ClN7OS Exact Mass: 539.2234 Molecular Weight: 540.1232
540.2


 


JQ42



   Chemical Formula: C23H25FN4O2S Exact Mass: 440.1682 Molecular Weight: 440.5336
441.2


 


JQ43



   Chemical Formula: C24H24ClN7OS Exact Mass: 493.1452 Molecular Weight: 494.0117
494.1


 


JQ44



   Chemical Formula: C25H29ClN6O2S Exact Mass: 512.1761 Molecular Weight: 513.0548
513.2


 


JQ45



   Chemical Formula: C24H24ClN7OS Exact Mass: 493.1452 Molecular Weight: 494.0117
494.1


 


JQ46



   Chemical Formula: C25H31ClN6OS Exact Mass: 498.1969 Molecular Weight: 499.0712
499.2


 


JQ47



   Chemical Formula: C32H44ClN7O2S Exact Mass: 625.2966 Molecular Weight: 626.2555
626.3


 


JQ48



   Exact Mass: 470.1543 Molecular Weight: 471.0148
471.2


 


JQ49



   Exact Mass: 428.1074 Molecular Weight: 428.9350
429.1


 


JQ50



   Exact Mass: 539.2234 Molecular Weight: 540.1232
540.2


 


JQ51



   JQI-II-114 Exact Mass: 666.1816 Molecular Weight: 667.1764
667.2


 


JQ52



   Exact Mass: 512.2125 Molecular Weight: 513.0978
513.2


 


JQ53



   Exact Mass: 399.1284 Molecular Weight: 399.9402
400.1





Spectral data for each compound were consistent with the assigned structure.






II. Biological Activity and Methods of Treatment
Example 1
JQ1 is an Inhibitor of BRDT
The feasibility of targeting human bromodomains with acetyl-lysine competitive small molecules was recently established (Filippakopoulos et al., Nature 468:1067 (2010)). The index study identified a potent thienodiazepine inhibitor ((+)-JQ1; FIG. 2A; Kd=90 nM) of the BET family co-activator protein BRD4 (Filippakopoulos et al.), which is implicated in the pathogenesis of cancer. Protein sequence alignment of human BRD4(1) to human BRDT(1) reveals 81% identity and 89% similarity, including all surface residues predicted to contact (+)-JQ1 (FIGS. 1 and 3). Based on these insights and preliminary evidence of binding to BRDT(1) established by differential scanning fluorimetry (Filippakopoulos et al.), the biochemical and functional effects of (+)-JQ1 on BRDT(1) were evaluated.
To assess competitive binding to BRDT(1), a homogeneous, luminescence proximity assay (alpha-screen), capable of quantifying binding of a synthetic, biotinylated tetra-acetylated histone 4 peptide (H4Kac4, residues 1-20) to recombinant epitope-tagged BRDT(1) was employed. Dose-ranging studies of (+)-JQ1 demonstrated potent inhibition of H4Kac4 binding, with a half-maximum inhibitory concentration (IC50) value of 11 nM (FIG. 2B). In contrast, the (−)-JQ1 stereoisomer was inactive for BRDT(1), establishing a stereospecific, ligand-competitive binding event.
Example 2
JQ1 Inhibits BRDT Activity During Spermatogenesis
To determine the possible consequences of blocking BRDT function in vivo, the spermatogenic effects of JQ1 administered to male mice were evaluated. Murine BRDT(1) exhibits 90% amino acid sequence identity and 95% similarity to human BRDT(1), including all surface residues influencing molecular recognition (FIG. 4), supporting the validity of using JQ1 in murine model systems. Juvenile or adult C57BL6/J/129S5 hybrid male mice were administered daily intraperitoneal injections of JQ1 (50 mg/kg/day) or vehicle control over a 3- or 6-week period. After 3 weeks of treatment, mice were either sacrificed or mated to females while continuing to receive JQ1. The JQ1-treated males universally were observed to have grossly smaller testes compared to the control males (FIG. 5A). At each time point, males treated with JQ1 experienced a marked and significant reduction in testes volumes (FIG. 5B). Males treated from 3-6 weeks of age showed a reduction to 75.4% of control, males treated from 6-9 weeks of age showed a reduction to 54.7% of controls, and males treated for 6 weeks with JQ1 (6-12 weeks of age) showed the most dramatic reduction to 40.6% of the controls (FIG. 5B). Consistent with the reduction in testes volumes, the tubules of JQ1 treated males were narrower with a decrease in the amount and number of tubules that had obvious and abundant spermatozoa in their lumen (FIGS. 5C-5F). Whereas an abundance of seminiferous tubules from the control mice were observed to be full of spermatozoa (FIGS. 5C and 5E), the number of tubules with spermatozoa and the amount of spermatozoa in these tubules were reduced in the JQ1-treated males (FIGS. 5D and 5F). Consistent with the reduction in testes weights (FIG. 5), the most dramatic findings in the JQ1-treated males (6 weeks treatment) were seminiferous tubule degeneration where few tubules contained significant numbers of mature spermatozoa (FIGS. 5E and 5G). Histological analysis of the epididymides of JQ1-treated males also showed a similar finding in which fewer sperm were observed in the epididymal lumen compared to the abundance observed in the control (FIGS. 5G and 5H). These results are consistent with the findings from a repeat study in which C57B6 mice were treated with JQ1 (FIGS. 8A-8C)
To further characterize these defects, spermatozoa number was determined after 3 weeks of treatment (3-6 weeks of age). It was found that epididymal sperm number were reduced to 27.8% of the control while after 6 weeks of treatment, the sperm in the cauda epididymis of the JQ1-treated mice were 10.9% of the control (FIG. 6A). Furthermore, whereas 85% of the sperm from the cauda epididymis of the control showed progressive motility, JQ1 treatment resulted in only 5% of the spermatozoa with progressive motility. Thus, JQ1 treatment quantitatively reduced sperm number and qualitatively reduced sperm motility. These findings phenocopy those observed in mice deficient in BRDT(1) (Shang et al., Development 134:3507 (2007)). Furthermore, the testosterone producing intertubular Leydig cells of the testes of JQ1-treated males appeared to be histologically normal (FIG. 6), and there appeared to be no defects in androgen actions in these mice since the testosterone-responsive seminal vesicles of JQ1-treated males were grossly normal. Lastly, since JQ1 had a significant effect on the seminiferous tubule compartment, it must be capable of effectively crossing the blood:testis boundary to alter spermatogenesis.
Example 3
JQ1 is a Reversible Inhibitor of BRDT Activity
To further evaluate the consequences of JQ1 on male fertility and fertilization potential, control (n=2) and JQ1-treated (n=3) males treated for 3 weeks were housed with 2 females each and subjected to treatments for an additional 3 weeks. Whereas the control males impregnated all 4 females, JQ1 had a contraceptive effect on the males (one failed to impregnate the two females, whereas only 1 of 2 females in each of the other two cages became pregnant). When these same males were test bred to superovulated females (2 females per cage), after 5 weeks of treatment, all females demonstrated copulation plugs indicating that JQ1 did not alter mating behavior, consistent with normal testosterone-responsive tissues in these males. Oocytes from these females were collected from their oviductal ampulla and cultured for 2 days to determine their developmental potential post-mating (FIG. 6B). Whereas the majority of the oocytes from females mated to controls developed into 2 cell (72.8%) and 4 cell (70.1%) embryos, few of the oocytes from the females mated to JQ1 males developed into 2 cell (10.1%) or 4 cell (6.6%) embryos, consistent with their lower sperm counts, decreased motility, and fertility defects. Importantly, the effects of JQ1 on male fertility were found to be reversible. Following cessation of JQ1, 6 of 6 JQ1-treated adult male mice sired two litters of offspring (7.25+/−0.58 pups per litter) within the first ensuing month. These results are consistent with the findings that the sperm motility, testes weight, and sperm count in male mice returned towards normal levels after cessation of JQ1 treatment (FIGS. 9A-9C).
Example 4
Molecular Analysis of JQ1 Mediated BRDT Inhibition
To molecularly define the stages of spermatogenesis at which JQ1 functions, quantitative RT-PCR was performed on testes isolated from JQ1-treated mice and controls (FIG. 7A). Genes expressed early in spermatogenesis such as Plzf, which is a marker for spermatogonial stem cells and early dividing spermatogonia (Buaas et al., Nat. Genet. 36:647 (2004); and Costoya et al., Nat. Genet. 36:653 (2004)), and Stra8, which is expressed mainly in differentiating spermatogonia and preleptotene spermatocytes (Zhou et al., Biol. Reprod. 79:35 (2008)), are 2.0-fold and 1.3-fold enriched, respectively, in the testes of JQ1-treated mice compared to control males. However, genes expressed during meiosis or spermiogenesis including Brdt (expressed in mid- to late-spermatocytes) (Shang et al., Gene Expr. Patterns 4:513 (2004)), Ccna1 (expressed in pachytene spermatocytes) (Sweeney et al., Development 122:53 (1996)), Papolb (expressed in step 1-7 round spermatids) (Kashiwabara et al., Dev. Biol. 228:106 (2000)), Klf17 (expressed in step 4-7 spermatids) (Yan et al., Mech. Dev. 118:233 (2002)), and Prm1 (expressed in step 7-16 spermatids) (Kleene et al., Dev. Biol. 105:71 (1984)) are 2.1-fold to 4.0-fold lower in the testes of mice treated with JQ1 versus control. Unlike the Brdt knockout studies (Shang et al., Development 134:3507 (2007)) in which the pachytene spermatocyte-expressed gene, Hist1h1t, is upregulated, JQ1 treatment leads to a 2.6-fold downregulation of this gene in line with the suppression of Ccna1. Consistent with these mRNA findings and the histological analysis described above, JQ1 treatment reduced the number of spermatids positive for transition protein 1 (TNP1) (FIGS. 7B and 7C), which is expressed in the nuclei of step 10-15 spermatids (Zhao et al., Biol. Reprod. 71:1016 (2004)).
A pharmacologic approach to male contraception remains a longstanding challenge in medicine. The results described herein provide pharmacologic validation of the amino-terminal bromodomain of BRDT as a target for male contraception, using a highly potent and selective chemical probe. JQ1 emerges as a lead compound for a new class of drugs that can cross the blood:testis boundary, inhibit bromodomain activity during spermatogenesis, impair sperm generation and motility, reduce the number of oocytes fertilized, and produce a reversible contraceptive effect in mammals. As human and mouse BRDT proteins are highly conserved and have nearly identical bromodomain pockets based on our structural predictions, these discoveries can be completely translated to men, and provide a novel and efficacious strategy for a male contraceptive.
The results reported herein were obtained using the following methods and materials.
(+)-JQ1 
The direct-acting, small-molecule bromodomain inhibitor was prepared as previously described (Filippakopoulos et al., Nature 468:1067 (2010)).
Protein Cloning, Expression and Purification
The N-terminal domain of human BRDT was cloned, expressed in E-Coli and purified as previously described (Filippakopoulos et al.).
BRDT Proximity Assay
Assays were performed with minor modifications from the manufacturer's protocol (PerkinElmer, USA). All reagents 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 to room temperature prior to addition to plates. After addition of Alpha beads to master solutions all subsequent steps were performed in low light conditions. A 2× solution of components with final concentrations of BRDT at 80 nM, Ni-coated Acceptor Bead at 25 μg/ml, and 80 nM biotinylated H4-tetra acetyl was added in 10 μL to 384-well plates (AlphaPlate-384, PerkinElmer, USA). Biotinylated peptide for BRDT was synthesized in-house on a CEM Liberty 9008005 microwave peptide synthesizer: H4-tetra acetyl, Biotin-PEG2-SGRGKacGGKacGLGKacGGAKacRHRK—COOH. Addition to wells was performed with either a multichannel pipet (for optimization experiments) or a Biotek EL406 liquid handler. After a 1 minute 1000 rpm spin-down, 100 nl of compounds from stock plates were added by pin transfer using a Janus Workstation (PerkinElmer, USA). The streptavidin-coated donor beads (25 μg/ml final) were added as with previous solution in a 2×, 10 μl volume. Following this addition, the plates were sealed with foil to block light exposure and to prevent evaporation. The plates were spun down again at 1000 rpm for 1 minute. Next, the plates were incubated in the room with the plate reader (for temperature equilibration) for 1.5 hour prior to reading the assay. AlphaScreen measurements were performed on an Envision 2104 (PerkinElmer, USA) utilizing the manufacturer's protocol.
Sequence Alignment
Amino acid sequences for full-length bromodomain-containing proteins were obtained from the US National Heart, Lung and Blood Institute (Human BRDT accession number Q58F21; Human BRD4 accession number 060885; Mouse BRDT accession number Q91Y44). Multiple sequence alignments of full-length BRDT and BRD4 were generated using MAFFT (v6.240) (Katoh et al., Nucleic Acids Res. 33:511 (2005); Katoh et al., Nucleic Acids Res. 30:3059 (2002); and Katoh and Toh, Brief Bioinform. 9:286 (2008)). The E-INS-i algorithm was selected as suitable for sequences containing potentially large unalignable regions, and the BLOSUM62 scoring matrix was used as suitable for highly evolutionarily conserved sequences. Gap opening penalty and offset value were set to default parameters.
Mouse Studies
(+)-JQ1 was dissolved in DMSO at 50 mg/ml and then diluted 1:10 in (2-Hydroxypropyl)-β-cyclodextrin (Sigma-Aldrich, St. Louis, Mo.). The subsequent mixture was injected intraperitoneal into male mice at 1% of the body weight of the mouse (final amount is 50 mg/kg/day). The control was DMSO dissolved 1:10 in (2-Hydroxypropyl)-β-cyclodextrin and injected similarly. Juvenile or adult C57BL6/J/12955 hybrid mice for these studies were weighed daily before injections and fed ad libitum. These studies were approved by the Administrative Committee on Laboratory Animal Care at Baylor College of Medicine, and all experiments were conducted in accordance with the NIH guide for the Care and Use of Laboratory Animals.
Histological Analysis
Histological analysis of Bouin's fixed testes and epididymides was performed as previously described (Kumar et al., Nature Genetics 15:201 (1997)) using Periodic acid-Schiff and hematoxylin. Rabbit anti-TNP2 (1:600) staining and hematoxylin counter-staining was performed as described (Zhao et al., Biol. Reprod. 71:1016 (2004)) using Bouin's fixed testes.
Epididymal Sperm Counts
Counts were performed on spermatozoa isolated from the entire epididymis or from the caudal epididymis of adult mice as described (Roy et al., Faseb J. 21:1013 (2007)). In brief, epididymides were dissected and placed in prewarmed M2 medium, minced, and incubated at 37° C. in a CO2 incubator prior to counting.
Fertilization and Embryo Developmental Potential
To evaluate the ability of spermatozoa of treated mice to mate with females and fertilize oocytes, 21-day-old C57BL6/J/129S5 hybrid females were injected with 5 IU of pregnant mare serum gonadotropin (PMSG; Calbiochem, EMD, Gibbstown, N.J.) followed by 5 IU of human chorionic gonadotropin (hCG; Calbiochem, EMD, Gibbstown, N.J.) 48 hours later and mated to treated males. Oocytes were isolated from ampullas of oviducts of females with copulation plugs, counted, and cultured in M16 medium (Sigma-Aldrich, St. Louis, Mo.) for 24 hours (for counting of 2 cell embryos) and 48 hours (for counting of 4 cell embryos) as described (Andreu-Vieyra et al., PLoS Biol. 8:e1000453 (2010); and Burns et al., Science 300: 633 (2003)).
Quantitative RT-PCR Analysis
Total RNAs from mouse testes were isolated using TRIzol reagent (Invitrogen, Carlsbad, Calif.). Total RNA was then reversely transcribed using Superscript III reverse transcriptase (Invitrogen, Carlsbad, Calif.). Quantitative PCR was performed using SYBR green master mix and customized primers (Table 1).







TABLE 1 







Primers for quantitative PCR









Gene




name
Forward
Reverse





Plzf
TGGAGAAGCATTTGGGTATCTACTC
AAGACGGCATGCTCAACACA



(SEQ ID NO: 5)
(SEQ ID NO: 6)


 


Stra8
GAGTGAGGCCCAGCATATGTC
CCTCTGGATTTTCTGAGTTGCA



(SEQ ID NO: 7)
(SEQ ID NO: 8)


 


Brdt
GCTTTGGGACTCCACAACTACTATG
GATTGTCCATTTTCCCCTTGATC



(SEQ ID NO: 9)
(SEQ ID NO: 10)


 


Ccna1
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 ID NO: 15)
(SEQ ID NO: 16)


 


Klf17
CCTCCCGTTTGTTCTCAACTTG
GGTGCATAGCCTGTTCCTTATTG



(SEQ ID NO: 17)
(SEQ ID NO: 18)


 


Prm1
TGCACAGAATAGCAAGTCCATCA
TGTGGCGAGATGCTCTTGAA



(SEQ ID NO: 19)
(SEQ ID NO: 20)









All quantitative PCR assays were conducted in duplicate for each sample. Gapdh was used as an internal control for the quantification.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. The subject matter described herein may be related to subject matter of U.S. provisional applications 61/334,991, 61/370,745, and 61/375,663, each of which is incorporated herein by this reference.

## Claims

1. A method of reducing or inhibiting spermatogenesis in a healthy fertile male subject in need thereof, the method comprising administering an amount of a compound represented by structural formula (I) sufficient to induce at least one of azoospermia, oligozoospermia, or asthenozoospermia:




wherein
X is N or CR5;
R5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
RB is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R3, each of which is optionally substituted;
ring A is aryl or heteroaryl;
each RA is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two RA together with the atoms to which each is attached, can form a fused aryl or heteroaryl group;
R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
R1 is —(CH2)n-L, in which n is 0-3 and L is —COO—R3, —CO—R3, —CO—N(R3R4), —S(O)2—R3, —S(O)2—N(R3R4), N(R3R4), N(R4)C(O)R3, optionally substituted aryl, or optionally substituted heteroaryl;
R2 is H, D, (deuterium), halogen, or optionally substituted alkyl;
each R3 is independently selected from the group consisting of:
(i) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
(ii) heterocycloalkyl or substituted heterocycloalkyl;
(iii) —C1-C8 alkyl, —C2-C8 alkenyl or —C2-C8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C3-C12 cycloalkyl, substituted —C3-C12 cycloalkyl, —C3-C12 cycloalkenyl, or substituted —C3-C12 cycloalkenyl, each of which may be optionally substituted; and
(iv) NH2, N═CR4R6;
each R4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
or R3 and R4 are taken together with the nitrogen atom to which they are attached to form a 4-10-membered ring;
R6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R4 and R6 are taken together with the carbon atom to which they are attached to form a 4-10-membered ring;
m is 0, 1, 2, or 3;

provided that
(a) if ring A is thienyl, X is N, R is phenyl or substituted phenyl, R2 is H, RB is methyl, and R1 is —(CH2)n-L, in which n is 1 and L is —CO—N(R3R4), then R3 and R4 are not taken together with nitrogen atom to which they are attached to form a morpholino ring;
(b) if ring A is thienyl, X is N, R is substituted phenyl, R2 is H, RB is methyl, and R1 is —(CH2)n-L, in which n is 1 and L is —CO—N(R3R4), and one of R3 and R4 is H, then the other of R3 and R4 is not methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl; and
(c) if ring A is thienyl, X is N, R is substituted phenyl, R2 is H, RB is methyl, and R1 is —(CH2)n-L, in which n is 1 and L is —COO—R3 is not methyl or ethyl,
or a pharmaceutically acceptable salt thereof,
thereby inducing a contraceptive effect in the healthy fertile male subject.

2. The method of claim 1, wherein the compound or a salt thereof is administered to the subject orally, transdermally, or by injection.

3. The method of claim 2, wherein the compound or a salt thereof is administered in the form of a tablet or capsule.

4. The method of claim 2, wherein the compound or a salt thereof is administered by parenteral injection, intramuscular injection, intravenous injection, subcutaneous implantation, subcutaneous injection, or transdermal preparation.

5. The method of claim 1, wherein the compound or a salt thereof is administered in combination with a pharmaceutically acceptable carrier, excipient, or diluent.

6. The method of claim 1, wherein the subject is human.

7. The method of claim 1, wherein the compound is represented by structural formula IV:




wherein:
X is N or CR5;
R5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
RB is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R3, each of which is optionally substituted;
ring A is aryl or heteroaryl;
each RA is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two RA together with the atoms to which each is attached, can form a fused aryl or heteroaryl group;
R1 is —(CH2)n-L, in which n is 0-2 and L is —COO—R3, —CO—R3, —CO—N(R3R4), —S(O)2—R3, —S(O)2—N(R3R4), N(R3R4), N(R4)C(O)R3, optionally substituted aryl, or optionally substituted heteroaryl;
R2 is H, D, halogen, or optionally substituted alkyl;
each R3 is independently selected from the group consisting of:
(i) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
(ii) heterocycloalkyl or substituted heterocycloalkyl;
(iii) —C1-C8 alkyl, —C2-C8 alkenyl or —C2-C8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C3-C12 cycloalkyl, substituted —C3-C12 cycloalkyl, —C3-C12 cycloalkenyl, or substituted —C3-C12 cycloalkenyl, each of which may be optionally substituted; and
(iv) NH2, N═CR4R6;
each R4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
or R3 and R4 are taken together with the nitrogen atom to which they are attached to form a 4-10-membered ring;
R6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R4 and R6 are taken together with the carbon atom to which they are attached to form a 4-10-membered ring;
m is 0, 1, 2, or 3; or
a pharmaceutically acceptable salt thereof.

8. The method of claim 7, wherein
L is —COO—R3, optionally substituted aryl, or optionally substituted heteroaryl; and
R3 is —C1-C8 alkyl, which contains 0, 1, 2, or 3 heteroatoms selected from O, S, or N, and which may be optionally substituted.

9. The method of claim 1, wherein the compound is represented by structural formula II,




wherein
X is N or CR5;
R5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
RB is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R3, each of which is optionally substituted;
each RA is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two RA together with the atoms to which each is attached, can form a fused aryl or heteroaryl group;
R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
R′1 is —COO—R3, —CO—R3, optionally substituted aryl, or optionally substituted heteroaryl;
each R3 is independently selected from the group consisting of:
(i) H, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
(ii) heterocycloalkyl or substituted heterocycloalkyl;
(iii) —C1-C8 alkyl, —C2-C8 alkenyl or —C2-C8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C3-C12 cycloalkyl, substituted —C3-C12 cycloalkyl; —C3-C12 cycloalkenyl, or substituted —C3-C12 cycloalkenyl; each of which may be optionally substituted; and
m is 0, 1, 2, or 3;
or a pharmaceutically acceptable salt thereof.

10. The method of claim 9, wherein
R′1 is —COO—R3, optionally substituted aryl, or optionally substituted heteroaryl; and
R3 is —C1-C8 alkyl, which contains 0, 1, 2, or 3 heteroatoms selected from O, S, or N, and which may be optionally substituted.

11. The method of claim 10, wherein R′1 is —COO—R3, and R3 is methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, or t-butyl.

12. The method of claim 1, wherein the compound is represented by structural formula (III):




wherein:
X is N or CR5;
R5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
RB is H, alkyl, hydroxyl alkyl, haloalkyl, alkoxy, or —COO—R3, each of which is optionally substituted;
ring A is aryl or heteroaryl;
each RA is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two RA together with the atoms to which each is attached, can form a fused aryl or heteroaryl group;
R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
each R3 is independently selected from the group consisting of:
(i) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
(ii) heterocycloalkyl or substituted heterocycloalkyl;
(iii) —C1-C8 alkyl, —C2-C8 alkenyl or —C2-C8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C3-C12 cycloalkyl, substituted —C3-C12 cycloalkyl, —C3-C12 cycloalkenyl, or substituted —C3-C12 cycloalkenyl, each of which may be optionally substituted; and
(iv) NH2, N═CR4R6;
each R4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl; each of which is optionally substituted;
R6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; and
m is 0, 1, 2, or 3;
or a pharmaceutically acceptable salt thereof.

13. The method of claim 12, wherein R is an optionally substituted aryl or an optionally substituted heteroaryl.

14. The method of claim 13, wherein R is an optionally substituted phenyl or an optionally substituted pyridyl.

15. The method of claim 14, wherein R is p-Cl-phenyl, o-Cl-phenyl, m-Cl-phenyl, p-F-phenyl, o-F-phenyl, m-F-phenyl or pyridinyl.

16. The method of claim 12, wherein R3 is H, NH2, or N═CR4R6.

17. The method of claim 12, wherein R4 is H, an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, or an optionally substituted heteroaryl.

18. The method of claim 12, wherein R6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted.

19. The method of claim 1, wherein the compound is selected from the group consisting of:







or a pharmaceutically acceptable salt thereof.

20. The method of claim 1, wherein the amount of the compound of formula (I) administered is sufficient to lower spermatozoa concentration to not more than 3 million/mL.

21. The method of claim 20, wherein the spermatozoa concentration is not more than a concentration selected from 2 million/mL, 1 million/mL, 0.5 million/mL, 0.25 million/mL, or 0.1 million/mL.

22. The method of claim 1, wherein the amount of the compound of formula (I) administered is sufficient to induce at least one of azoospermia or oligozoospermia.