AU2006201538B2 - Selective androgen receptor modulators and methods of use thereof - Google Patents

Description

Cz- r 7 Va 0 0,

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant: University of Tennessee Research Foundation Invention Title: SELECTIVE ANDROGEN RECEPTOR MODULATORS AND METHODS OF USE THEREOF

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The following statement is a full description of this invention, including the best method of performing it known to me/us:

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SELECTIVE ANDROGEN RECEPTOR MODULATORS AND Cxl METHODS OF USE THEREOF s FIELD OF INVENTION 00 The present invention relates to a novel class of tissue-selective androgen t receptor targeting agents (ARTA) which demonstrate androgenic and anabolic activity Sof a nonsteroidal ligand for the androgen receptor. The agents define a new subclass of compounds which are tissue-selective androgen receptor modulators (SARM) which are So0 useful for male hormone therapy such as oral testosterone replacement therapy, male Scontraception, maintaining sexual desire in women, treating prostate cancer, and imaging prostate cancer. These agents are also administered to a subject for the treatment of sarcopcnia, lack of sexual libido, osteoporosis, erythropoiesis, and fertility.

The agents may be used alone or in combination with a progestin or estrogen.

BACKGROUND OF THE INVENTION The androgen receptor is a ligand-activated transcriptional regulatory protein that mediates induction of male sexual development and 'function through its activity with endogenous androgens. Androgens are generally known as the mate sex hormones. However, androgens also play a pivotal role in female physiology and reproduction. The andragenic hormones are steroids which are produced in the body by the testis and the cortex of the adrenal gland, or synthesized in the laboratory.

Androgenic steroids play an important role in many physiologic processes, including the development and maintenance of male sexual characteristics such as muscle and bone mass, prostate growth, spermatogenesis, and the male hair pattern (Matsumoto, SEndocrinol. Met. Clin. N. Am. 23:857-75 (1994). The endogenous steroidal androgens include testosterone and dihydrotestosrerone Testosterone is the principal steroid secreted by the testes and is the primary circulating androgen found in the plasma of males. Testosterone is converted to DHT by the enzyme 5 alpha-reduotase in many peripheral tissues. DHT is thus thought to serve as the intracellular mediator for most androgen actions (Zhou, et al.. Molec. Endocrinol. 9:208-18 (1995))- Other steroidal androgens include esters of testosterone, such as the cypionate, propionate, phenylpropionate, oyclopentylpropionate, isocarporate, enanthate, and decanoate esters, and other synthetic androgens such as 7-Methyl-Nortestosterone ("MENT") and its 0 0

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Sacetate ester (Sundaram et al., "7 Alpha-Methyl-Nortestosterone(MENT): The Optimal Androgen For Male Contraception," Ann. Med., 25:199-205 (1993) ("Sundaram")).

c' Because the AR is involved in male sexual development and fimunction, the AR is a likely target for effecting male contraception or other forms of hormone replacement therapy.

s The AR also regulates female sexual function libido), bone formation, and 00 ec ryhropoiesis.

C Worldwide.population growth and social awareness of family planning have o stimulated a great deal of research in contraception. Contraception is a difficult subject o 10 under any circumstances. It is fraught with cultural and social stigma, religious implications, and, most certainly, significant health concerns. This situation is only exacerbated when the subject focuses on male contraception. Despite the availability of suitable contraceptive devices, historically, society has looked to women to be responsible for contraceptive decisions and their consequences. Although health concerns over sexually transmitted diseases have made men more aware of the need to develop safe and responsible sexual habits, women still often bear the brunt of contraceptive choice. Women have a number of choices, from temporary mechanical devices such as sponges and diaphragms to temporary chemical devices such as spermnnicides. Women also have at their disposal more permanent options, such as physical devices like IUDs and cervical caps as well as more permanent chemical treatments, such as birth control pills and subcutaneous implants. However, to date, the only options available for men include the use of condoms or a vasectomy. Condom use, however is not favored by many men because of the redceod sexual sensitivity, the interruption in sexual spontaneity, and the significant possibility of pregnancy caused by breakage or misuse. Vasectomies are also not favored. If more convenient methods of birth control were available to men, particularly long term methods that require no preparative activity immediately prior to a sexual act, such methods could significantly increase the likelihood that men would take more responsibility for contraception.

Administration of the male sex steroids testosterone and its derivatives) has shown particular promise in this regard due to the combined gonadotropinsuppressing and androgen-substituting properties of these compounds (Steinberger et al.,

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S"Effect of Chronic Administration of Testosterone Enanthate on Sperm Production and Plasma Testosterone, Follicle Stimulating Hormone, and Luteinizing Hormone Levels: A Preliminary Evaluation of a Possible Male Contraceptive", Fertility and Sterility 28:1320- 28 (1977)). Chronic administration of high doses of testosterone completely s abolishes sperm production (azoospernnia) or reduces it to a very low level 00 Cfr (oligospermia). The degree of spermatogenic suppression necessary to produce infertility is not precisely known. However, a recent report by the World Health Organization C showed that weekly intramuscular injections of testosterone enanthate result in I azoospermia or severe oligospermia loss than 3 million sperm per ml) and infertility T 1o in 98% of men receiving therapy (World Health Organization Task Force on Methods Ar Regulation of Male Fertility, "Contraceptive Efficacy of Testosterone-Induced Azoospennia and Oligospermia in Normal Men," Fertilily and Sterility 65:821-29 (1996)).

A variety of testosterone esters have been developed that are more slowly absorbed after intramuscular injection and, thus, result in greater androgenic effect.

Testosterone enanthate is the most widely used of these eaters. While testosterone enanthate has been valuable in terms of establishing the feasibility of hormonal agents for male contraception, it has several drawbacks, including the need for weekly injections and the presence of supraphysiologic peak levels of testosterone immediately following intramuscular injection (Wu, "Effects of Testosterone Enanthate in Normal Men: Experience Prom a Multicenter Contraceptive Efficacy Study," Fertility and Sterility 65:626-36 (1996)).

2s Steroidal ligands which bind the AR and act as androgens testosterone enanthate) or as antiandrogens cyproterone acetate) have been known for many years and are used clinically (Wu 1988). Although nonsteroidal antiandrogens are in clinical use for hormone-dependent prostate cancer, nonsteroidal androgens have not been reported. For this reason, research on male contraceptives has focused solely on steroidal compounds.

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0 k SUMMARY OF THE INVENTION C-I This invention provides a novel class of tissue-selective androgen receptor targeting agents (ARTA). The agents define a new subclass of compounds which are s tissue-selective androgen receptor modulators (SARM), which are useful for oral 00 (cr testosterone replacement therapy, male contraception, maintaining sexual desire in women, osteoporosis, treating prostate cancer and imaging prostate cancer. These C agents have an unexpected and tissue-selective in-vivo activity for an androgenic and o anabolic activity of a nonsteroidal ligand for the AR. These agents selectively act as S 10 partial agonists in some tissues, while acting as full agonists in other tissues, providing a a novel and unexpected means for eliciting tissue-selective androgenic or anabolic effects. These agents may be active alone or in combination with progestins or cstrogens. The invention further provides a novel class of non-steroidal agonist compounds. The invention further provides compositions containing the selective 1l androgen modulator compounds or the non-steroidal agonist compounds and methods of binding an AR, modulating spennatogenesis, bone formation and/or resorption, treating and imaging prostate cancer, and providing hormonal therapy for androgen-dependent conditions.

The present invention relates to a selective androgen receptor modulator compound having tissue-selective in-vivo androgenic and anabolic activity of a nonsteroidat ligand for the androgen receptor, the selective androgen receptor modulator compound represented by the structure of formula I: Va 0 wherein X is a o. CH 2 NH, So, PR, or NR, C- Z is NO 2 ON, COP, COOH or CONHR; V is L CE 3 Br, Cl, or SnR 3 Q is alkyl, halogen, Ni, NHCOCH 3

NHCOCF

3 NHCOR, NHCONHR, 00 cc N{COOR OCONHI CONHI, NWCSCH 3

NHOSCE

3 NHCSP, NHSOCHI, NUSOzK OR, CO& OCOk OSOtR, SO 2 R or SR 0~ wherein K is a alkyl, ary, hydroxy, CI-C alkyl, a CI-C4 haloalky], phony!, halo, alkenyl or hycroxyl; or Q together with the beuzene ring to which it is anached is a fused ring system represented by structure A. B or C: 0 cc A

C

Is K 1 is CH3, CFa. CIs. or CPCF,; and T is OH, OP, -NHCOC* 3 or NECOR wherein R is a I-C 4 alkyl, a C I- 04 haloalkyl, phenyl, halo, elkenyl or hydroxyl.

In one emnbodiment, Q is in the para position. In another embodiment, X is 0.

In another embodhnent, Q is in the pam position and X is 0. In yet another embodiment, Q is pars alkyL, halogen, Ni 2 NHCOCH3, NHCOCMj, NHCOR.

NHCONHR NHCOOU, OCONHR, CONH NHCSCH 3

NECSCF

3

NHCSR

NHShCH 3 NN801 OR, CO, OCOk, 0SOP, SR or SR wherein R is 4 alkyl, ary), hydroxy, Cj-C4 alkyL, a CI-C 4 haloalkyl, phonyl, halo, alkenyl OT hydroxyl.

2S The present Invention relates to a selective androgen receptor modulator compound having in-vivo anidrogenio and anabolic activity of a nonsteroldal ligand for 07/02/2008 07/022000 13:07 GRIFFITH HACK 4 IP AUSTRA~LIA PTNO46 NO.34G woe 00 0 rn INOa wherin Xis Q Z0sa0dg od=",N2 N OCNR Y salpdsoulgop R_ isaly1o rlgop rOi n Qi is aehydoe bafu aceto, O 2 CamN, er, ONHR -uloyl -slbal alkylsufonyl or waboalyl.

The pmurn inventio also atos toa scadte MWukoM recptr m daor cenapoqu hevlig iM-IVO mnropuic m4 ausble actt of IL mmatmldalW ligad ftr IS thc W*Mad n capt Uk lclctvo andnga rmcptormwoftokt ompmn repunjd by fts ihuct of fbqnul IM whew X IsaO0 ,C NK SkPL orMR; Z In NO, 0 COOP or CONElt Y it, CBr, q or Wht; R Ir. anklt or gr4rA pa (MI. and Q ix atamido, cc uifluroaaobsdo The react Irftic so Matom to a mekotli audromi tcqsnr modulator ,;ompsnl bxits h.jwl-vi wlrceua mid ausboll actvity of a 6 COMS ID No: ARCS-i 78323 Received by IP Australia: lime 14:03 Date 2008-02-07

I

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0 00 nonsteroidal ligad for the androgen receptor, the selective androgen receptor modulator compound represented by the stucture of formula IV:

O

N

0 ~CF N3 NHO F 11 C OH 0 0

IV

The present invention also relates to a selective androgen receptor modulator compound having tissue-selective in-vivo androgenic and anabolic activity of a nonsteroidal ligand for the androgen receptor, the selective androgen receptor modulator o compound represented by the structure of formula V: 02N COCH2

O

CP~' NHO

H

3

OH

V

The present invention also relates to a selective androgen receptor modulator compound having tissue-selective in-vive androgenic and anabolic activity of a Is nonstoroldal ligand flr the androgen receptor 1 the selective androgen receptor modulator compound represented by the structure of formula VI: 0 2 N COCR2CH CFs H

VI

J I Va 0 0 ci The present invention also relates to a selective androgen receptor modulator compound having tissue-selective in-vivo androgenic and anabolic activity of a nonsteroidal ligand for the androgen receptor, the selective androgen receptor modulator O0 5 compound represented by the structure of formula VII:

M

tn o ,N O N H

COCH

IND

Va* I~a" N ^t^

CF

3 H-b 011

VII

The present invention also relates to a method of binding a selective androgen receptor modulator compound to an androgen receptor, which includes contacting the androgen receptor with the selective androgen receptor modulator oompound under conditions effective to bind the selective androgen receptor modulator compound to the androgen receptor. In one embodiment the compound is Compound I. In another Is embodiment the compound is Compound 1. In another embodiment the compound is Compound Mi. In another embodiment the compound is Compound IV. In another embodiment the compound is Compound V. In another embodiment the compound is Compound VI. In another embodiment the compound is Compound VIH- In another embodiment the compound is Compound VIl.

Another aspect of the present invention relates to a method of modulating spermatogenesis in a subject, which includes contacting an androgon receptor of the subject with a selective androgen receptor modulator compoimd under coniditions effective to increase or decrease sperm production. In one embodiment the compound is Compound I. In another embodiment the compound is Compound I. In another embodiment the compound is Compound I. In another embodiment the compound is Compound IV. In another embodiment the compound is Compound V. In another

I

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0 Sembodiment the compound is Compound VI. In another embodiment the compound is Compound VII. In another embodiment the compound is Compound VIII.

The present invention also relates to a method of hormone therapy, comprising contacting an androgen receptor of a subject with a selective androgen receptor 00 cc modulator compound under conditions effective to bind the selective androgen receptor tt modulator compound to the androgen receptor and effect a change in an androgen- 0 dependent condition. In one embodiment the compound is Compound I. In another Sembodiment the compound is Compound I. In another embodiment the compound is Sto Compound m. In another embodiment the compound is Compound IV. In another embodiment the compound is Compound V. In another embodiment the compound is Compound VI, In another embodiment the compound is Compound VII. In another embodiment the compound is Compound VII- The present invention also relates to a method of treating a subject having a hormone related condition which comprises contacting an androgen receptor of said subject with a selective androgen receptor modulator compound under conditions effective to bind the selective androgen receptor modulator compound to the androgen receptor and effect a change in an androgen-dependent condition. In one embodiment, the selective androgen receptor modulator compound is selective for androgen or testosterone receptor. The present invention also relates to a method of oral administration of the selective androgen receptor modulator compound.

The present invention also relates to a method of treating a subject having a chronic muscle wasting disease state which comprises contacting an androgen receptor of said subject with a selective androgen receptor modulator compound as described herein under conditions effective to bind the selective androgen receptor modulator compound to the androgen receptor and effect a change in an androgen-dependent condition. In one embodiment, the selective androgen receptor modulator compound is selective for androgen or testosterone receptor. The present invention also relates to a method of oral administration of the selective androgen receptor modulator compound.

In one embodiment the compound is Compound I. In another embodiment the

L

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0 0 Scompound is Compound 11. In another embodiment the compound is Compound IL. In another embodiment the compound is Compound IV. In another embodiment the C-i compound is Compound V. In another embodiment the compound is Compound VI. In another embodiment the compound is Compound VII. In another embodiment the compound is Compound VII.

In SAs defined herein, the disease state of "chronic muscle wasting" means 0 o The present invention also relates to a method of treating a subject having C 10o prostate cancer which comprises administering to a subject an effective amount of a selective androgen receptor modulator compound. In one embodiment, the selective androgen receptor modulator compound is selective for androgen or testosterone receptor. In one embodiment the compound is Compound 1. In another embodiment the compound is Compound II. In another embodiment the compound is Compound Ill. In Is another embodiment the compound is Compound IV. In another embodiment the compound is Compound V. In another embodiment the compound is Compound VI. In another embodiment the compound is Compound VII. In another embodiment the compound is Compound VIl.

The present invention also relates to compositions and a pharmaceutical compositions which comprises a selective androgen receptor modulator alone or in combination with a progestin or estrogen and a suitable carier, diluent or salt. In one embodiment the composition comprises Compound I. In another embodiment the compound is Compound IL In another embodiment the compound is Compound I. In another embodiment the compound is Compound IV. In another embodiment the compound is Compound V. In another embodiment the compound is Compound V1. In another embodiment the compound is Compound VII. In another embodiment the compound is Compound VIII.

The present invention relates to a non-steroidal agonist compound, the nonsteroidal agonist compound represented by the sructure of formula VIII: 0 NH X Ri T VIll Va 0 0, wherin Xis sO, CH 2 NHtS; PR, orNR; R, is CH,, CE,, CH 2 or CEa2C?, T is OR, OR, -NHCOCH-,, or NHCOR wherein R is a C,-C 4 alkyl, a CI- C4 haloalkyl, phcnyl. halo, alknyl or hydroxyl: A Is a 5 or 6 membered saturated, unsaturnted or aromatic carbocyclic or beterocyclic ring represented by the structure: Z A 2 A A 1 or As...

Ag

AC

AiW./ S is a 5 or 6 membered saurated, imsaturuted or aromatic carbocyclic or heterocyclic ring represented by the stnocture: or /s- BI.. B Qz Dl CiQiI wherein At- Ail am each C,O, S orN; B B, are each C, 0, S or N; Z is N2, CN. COOM COP, or CQNRR Y is I, CP3, Sr, CI, or StIR,; and Qi and Q are independently of each other alkyl. halogen, NR., NHCOCM,, NECOCF3, NUCOR, NHCONH&t NNCOOR, OCONHR. CONHR, NHCSCH, NHCSCF,, NHCSR NHSQ 2 CH,, NMSO 2 P, OR, CO. OCOR, OSOz, SO2R or SR wherein R is a C,-C 4 alkyl, n C 1 haloalkyl, phenyl, hIo, alkenyl or hydroxyl.

I I

ID

0 0

(N

_The present invention also relates to a composition and pharmaceutical composition comprising the non-steroidal agonist compound alone or in combination c-i with a progeatin or estrogen and a suitable carrier, diluent or salt. In one embodiment the compound is Compound I. In another embodiment the compound is Compound I1. In 5 another embodiment the compound is Compound IU. In another embodiment the 00 compound is Compound IV. In another embodiment the compound is Compound V. In another embodiment the compound is Compound VI. In another embodiment the 0' compound is Compound VII. In another embodiment the compound is Compound VIII.

c0 0 The present invention also relates to a rmethod of binding a non-steroidal agonist compound to an androgen receptor comprising contacting the androgen receptor with the non-steroidal agonist compound under conditions effective to bind the nonsteroidal agonist compound to the androgen receptor. In one embodiment the compound is Compound I. In another embodiment the compound is Compound 1. In another enmbodiment the compound is Compound III. In another embodiment the compound is Compound IV. In another embodiment the compound is Compound V. In another embodiment the compound is Compound VI. In another embodiment the compound is Compound VII. In another embodiment the compound is Compound VII.

The present invention also relates to a method of modulating spermnnatogenesis in a subject comprising contacting an androgen receptor of the subject with a nonsteroidal agonist compound under conditions effective to increase or decrease sperm production. In one embodiment the compound is Compound I. In another embodiment the compound is Compound U. In another embodiment the compound is Compound HI.

In another embodiment the compound is Compound IV. In another embodiment the compound is Compound V. In another embodiment the compound is Compound VI. In another embodiment the compound is Compound VU. In another embodiment the compound is Compound VIII.

The present invention also relates to a method of hormone therapy comprising contacting an androgen receptor of a subject with a non-steroidal agonist under conditions effetive to bind the nonr-steroidal agonist compound to the androgen receptor

ID

0 0 and effect a change in an androgen-dependent condition. In one embodiment the compound is Compound L In another embodiment the compound is Compound II. In ci another embodiment the compound is Compound III. In another embodiment the compound is Compound IV. In another embodiment the compound is Compound V. In another embodiment the compound is Compound VI. In another embodiment the 00 Scompound is Compound VII. In another embodiment the compound is Compound VIII.

o The present invention also relates to a method of treating a subject having a IND hormone related condition which comprises contacting an androgen receptor of said 0 10 subject with a non-steroidal agonist compound under conditions offective to bind the non-steroidal agonist compound to the androgen receptor and effect a change in an androgen-dependent condition. In one embodiment, the non-steroidal agonist compound is selective for androgen or testosterone receptor. The present invention also relates to a method of oral administration of the non-steroidal agonist compound. In one embodiment the compound is Compound I. In another embodiment the compound is Compound II. In another embodiment the compound is Compound III. In another embodiment the compound is Compound IV. In another embodiment the compound is Compound V. In another embodiment the compound is Compound VL In another embodiment the compound is Compound VII. In another embodiment the compound is Compound VIII.

The present invention also relates to a method of treating a subject having prostate cancer which comprises administrating to a subject an effective amount of a non-steroidal agonist compound. In one embodiment, the non-steroidal agonist compound is selective for androgen or testosterone receptor. In one embodiment the compound is Compound L In another embodiment the compound is Compound H. In another embodiment the compound is Compound II. In another embodiment the compound is Compound IV. In another embodiment the compound is Compound V. In another embodiment the compound is Compound VI. In another embodiment the compound is Compound VII. In another embodiment the compound is Compound VIII.

Still another aspect of the present relates to a method of producing a selective 0

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0 Sandrogen receptor modulator or a non- steroidal AR agonist compound of the present invention. In one embodiment the compound is Compound I. In another embodiment the compound is Compound II. In another embodiment the compound is Compound mI. In another embodiment the compound is Compound IV. In another embodiment the compound is Compound V. In another embodiment the compound is Compound VI. In 00 M another embodiment the compound is Compound VII. In another embodiment the compound is Compound VIII.

0 o, The present invention further relates to a method of determining the presence 0 to of a selective androgen modulator compound and/or a non-steroidal agonist compound of the present invention in a sample. The method comprises the steps of obtaining the sample, and detecting the compound in the sample, thereby determining the presence of the compound in the sample. In one embodiment the sample is a blood serum, plasma, urine, or saliva sample. In another embodiment, the detection step comprises measuring the absorbance of the compound. In one embodiment the compound is Compound I. In another embodiment the compound is Compound II. In another embodiment the compound is Compound I. In another embodiment the compound is Compound IV. In another embodiment the compound is Compound V. In another embodiment the compound is Compound VI. In another embodiment the compound is Compound VII. In another embodiment the compound is Compound VIII.

The novel selective androgen receptor modulator compounds and the nonsteroidal agonist compounds of the present invention, either alone or as a composition, are useful in males and females for the treatment of a variety of hormone-related conditions, suoh as hypogonadism, saroopenia, rythropoisis, eretile function, lak of libido, osteoporesis and fertility. Further, the selective androgen receptor modulator compounds and the non- steroidal agonist compounds are useful for oral testosterone replacement therapy, treating prostate cancer, imaging prostate cancer, and maintaing sexual desire in women. The agents may be used alone or in combination with a progestin or estrogen. In one embodiment the compound is Compound I. In another embodiment the compound is Compound II. In another embodiment the compound is Compound III. In another embodiment the compound Is Compound TV. In another

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embodiment the compound is Compound V. In another embodiment the compound is e- Compound VI. In another embodiment the compound is Compound VI. In another embodiment the compound is Compound VII.

s The selective androgen receptor modulator compounds and the non-steroidal

C

0 agonist compounds of the present invention offer a significant advance over steroidal In androgen treatment because the selective androgen receptor modulator compounds and O the non-steroidal agonist compounds of the present invention have been shown in-vivo \O to have a tissue-selective androgenic and anabolic activity of a nonsteroidal ligand for So the androgen receptor. Moreover, the selective androgen receptor modulator compounds and the non-steroidal agonist compounds of the present invention are not accompanied by serious side effects, lability to oxidative metabolism, inconvenient modes of administration, or high costs and still have tho advantages of oral bioavailability, lack of cross-rectivity with other steroid receptors, and long biological half-lives. In one embodiment the compound is Compound I. In another embodiment the compound is Compound II. In another embodiment the compound is Compound M. In another embodiment the compound is Compound IV. In another embodiment the compound is Compound V. In another embodiment the compound is Compound VI. In another embodiment the compound is Compound VII. In another embodiment the compound is Compound VII.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be understood and appreciated more flly from the following detailed description take in conjunction with the appended drawings in which: Figure 1; Androgenic and Anabolic activity of (S)-GTx-007 in rts. Rats were left untreaed (intact control), astrated (castrated control), treated with testosterone propionate or treated with S-GTx-007, and the body weight gain as well as the weight of androgen-responsive tissues (prostate, semimal vesicles and levator ani musole) was determined.

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0 Figure 2: Androgenic and Anabolic activity of S-GTx007 in rats. Rats were left untreated (inact control), castrated (castrated control), treated with 0.1, 0.3, 0.75 and 1.0 mg/day testosterone propionate or treated with 0.1, 0.3, 05, 0.75 and 1.0 mg/day S-GTx-007, and the weight of androgen-Tesponsive 00 Mtissues (prostate, semimal vesicles and levator ani muscle) was determined.

Figure 3: Androgenic and Anabolic activity of S-GTx-014 in rats. Rats were left oUntreated (intact control), castrated (castrated control), treated with 0.1. 0.3, o 0 0.5, 0.75 and 1.0 mg/day testosterone propionate CTP), or treated with 0.1, 0.3, 0.75 and 1.0 mg/day S-GTx-014, and the weight of androgen-responsive tissues (prostate, semimal vesicles and levator ani muscle) was determined.

Figure 4: Average plasma concentration-time profiles of S-GTx-007 in beagle dogs is after IV administration at 3 and 10 mg/kg.

Figure 5: Average plasma concentration-time profiles of S-GTx-007 in beagle dogs after PO administration as solution at 10 mglkg.

Fig 6: Avenge plasma concentration-time profiles of S-GTx-007 In boagle dogs after IV administration as vapsl1os at mg/kg.

Figure 7: Effects of GTx-014 and OTx-007 on LH Levels.

Figure 8: Effects of QTx-014 and GTx-007 on FSH Levels.

Figure 9: Synthesis scheme of 0TX-007.

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DETAILED DESCRIPTION OF THE INVENTION SThis invention provides a novel class of androgen reoeptor targeting agents (ARTA). The agents define a new subclass of compounds which ae tissue-selective s androgen receptor modulators (SARM) which are usefl for oral testosterone 0 replacement therapy, male contraception, mainaining sexual desire in women, treating prostate cancer and imaging prostate cancer. These agents have an unexpected tissue- O selective in-vivo activity for an androgenic and anabolic activity of a nonsteroida] ligand.

O for the androgen receptor. These agents may be active alone or in combination with Sto progestins or estrogens. The invention frther provides a novel class of non-steroidal agonist compounds. The invention further provides compositions containing the selective androgen modulator compounds or the non-steroidal agonist compounds and methods of binding an andrdgen receptor, modulating spermatogenesis, treating and imaging prostate cancer, and providing hormonal therapy for androgen-dependent conditions.

The compunds described herein, define a new class of selective androgep receptor modulators (SARMS) that demonstrate potent anabolic effects muscle growth) with less androgenic activity prostatic growth). This new class of drags has several advantages over non-selective androgens, including potential therapeutic applications in males and females for modulation of fertility, erythropoiesis, osteoporosis, sexual libido and in men with or at high risk for prostate cancer. In one embodiment the compound is Compound I. In another embodiment the compound is Compound I1. In another embodiment the compound is Compound E. In another embodiment the compound is Compound IV. In another embodiment the compound is Compound V. In another embodiment the compound is Compound VI. In another embodiment the compound is Compound VII In another embodiment the compound is Compound VIII.

Further, in one embodiment the compunds have tissue specific pharmacologic activity. As demonstarted in Figure 7 and 8. GTx-007 does not suppress LH levels at doses that arc capable of eliciting maximal stimulation of levator ani muscle growth and I i Va does not suppress FSH lewpis at doses that are capable of liciting mnlrnal sainulation oflevator ani muscle growh.

The present invention relates to a selective ndrogon receptor modulator compound having tissue-selective in-vivo androgenic and anabalic activity of a 00 M nonsteroldal ligand for the endrogen receptor selective androgen receptor modulator _compound represented by the structure of formula 1: c Va

I

wheztin X is a 0, CCz2 NH. Be, PR, or NR, Z is CN. COR, COON or CONHR; Jo Y is I, C 3 r, Cl, orSnR; Q is alkyl. halogen, NR 2 NUCOCH,, NECOCF,, NHCOR, NICONH NECOOR OCONIIR, CONIMR NHCSCH 3 NHCSCF,, NHCSR NHS02CIt3.

NHS

2 R OR, COR OCOR, OSO 2 R, SO 2 R or SR wherein R Is an ezyl. C 1

-C

4 alkyl 4 C 1 -C haloallyl, phenyl, halo. alkenyl or hydroxyl: or Q together with the bentene ring to which it is intched is a fused ring system represented by stncturc A, B or C: O 0 NH A C C 07/02/2008 07/022008 13:07 GRIFFITH HACK 4 IP AUSTRALIA PTN.38 Q0 NO.346 [POO9 00 0 o~mdm T is Oi, tOR PamOU 3 PostionflCis kywhai a12 OrCi akyl 3 s bxd R. uisyl phoy! ha4o u)Q fLkenylh~&. IMW or hyYnxyl.

Inq~ cuepboimn, Qp ii 1* o ass pwmon a thu am in t whch aWi embodimn Q is int0; npsto n .aly aoePk.NCC InNH~cL Z irgnHondapori, N02,GU ocoIR CONHZ NUCNcH, whRe Kissa ayl orC rlky arCohupl boy, ao aky or OH;rand Q is The juto trlaruaoreates m aaoiw etoplk. weeptfory 0uont is cmpond avlmnl tvo uroni admcolsatvt f os.od~ gu t C*ES sete. is slene ndomcveopormduatr omondrum9e COMS ID No: ARCS-i 78323 Received by IP Australia: Time 14:03 Date 2008-02-07

VO

0 The present invention also relates to a selective androgen receptor modulator compound having in-vivo androgeic and anabolic activity of a nonsteroidal ligand for the androgen roptor the, selective androgen receptor modulator compound represented by the structure of fonnula Ea: H 3 C

OH

00 CHzc Y U NN X cKI III

VO

o where X is a O, CH 2 NHSe,PRor NR; Ci Z is NO 2 CN, COR, -or CONER:; Y is I, CF., Br, Cl, or SnIa; R is an alkyl or aryl group r OH; and Q is acetamido or trifluroaetamido, l0 The present invention also relates to a selective androgen receptor modulator compound having tissue-selective in-vivo androgenic and anabolic activity of a nonsteroidal ligand for the androgen receptor, the selective androgen receptor modulatar compound represented by the structure of formula IV: C N

C

3 NH O

IV

The prsent invention also relates to a selective androgen receptor modulator compound having tissue-selective in-vive androgenic and anabolic activity of a ronsteroidal ligand for the androgen receptor, the selective androgen rceptor modulator compound represented by the structure of formula V:

I

\O

ON CCH3

OOO

3F3

NI,

V

The present invention also relates to a selective androgen receptor modulator

\O

O compound having tissue-salceive in-vivo androgenic and anabolic ctivity of B N~ S nonstaroidal ligand for the androgen receptor, the selective androgen receptor modulator compound represented by the structure of formula VT: 02N 00 CF NH

VI

The present invention also relates to a selective androgen receptor modulator compound having tissue-seloective in-vivo androgenic and anabolic activity of a co nonsteroidal ligand for the androgen receptor, the selective androgen receptor modulator compound represented by the structure of formula VH: ozN NHCOCH -O I C 2Z1 NH H i0 OH

VI

The present invention relates to a non-steromdal agoist ompound having the is formula (Compound Va):

I

Va 0 0, NH X Rl T

VEII

s wherein X is a O, CH. NN,Sa. ?Xtor NR; ft is CH 3

CF

3 C1 2 CH%, or CF 2

CF

3 T is OH, OR. -NHCOC43, or NHICOR wherein wherein R is a Cl-C, alkyl. a C 1

-C

4 halcafyl. pbanyL halo. slkayl or hydroxyl; A is a 5 or 6 membered ataatcd unsaturated or armatic carbocyclic or o hecrocyclic ring represenred by the strature: Z A 3

A

3 44 'At- or

A

z~ A16-- .A7 B is a 5 or 6 membered saturated, unsmatrated or aromatic carbocyclic or heterocyclic ring represented by the sructur: a: or

F

Sir 1 Va wherein All 1r each C, O, S or N; BI-E I ae each C, 0. S or N; 00 s Z is NO2, CN, COp, COC, or CONR; en Y is I, C3, Br, C1, or Sn&3; and Qi Q ad Q2 are independently of each other alkyl, halogen

N.

2 NICOCHL NNOCF, NHCOR, NHCONHR, NHCOoR, OCONRP,

CONHI,

NHCSCfIJ

NUCSCF

3 NHCSR bUSOaCH 3 NHSR, OR, COP, OCOR, to OSOJ( SOR or 8k wherein R is a C-C 4 alkyl, a Cj-C 4 hajoalkyl, phonyl, halo.

alkenyj or hydroxyl.

The substitqtents Z and Y can be in any position of the five or 6 membered ring carrying these mubstitotents (hereinfter "A ring"). Similarly, the substituon: Q can be in any position of the five or 6 membered ring carrying this substituten: (hereinafter

"B

ring"). It is Understood that when any of the ring members Al, orB 1 are 0 or S, then these ring member ar unsuhstitutcd. It is fbnaher understood that when any of the ring members AI or B 1

B

11 ar 0 or S, then toe doted line between said ring members and other ring members represerns a uingle bond.

In one embodimen, the A ring includes any ype of saturated or unsatnted Qarbocyclic ring. In one embodiment the A ring is a 6 membered saturated carbocyclic rng, which may be unsubstituted, monosubstitiaod or polysubstitutar by any of the substitutents deacn1od hereinabove. In one embodiment, the A ring is a 5 membered saturated curbocycjic ring, which may be unsubstituted monosubstituted or polysubstituted by any of the subatitutents described hereinabove. In another embodiment the A ring is a 6 membered carbocyulic ring containing one or more double bonds, which ring may be unsubstituted, monosubsdtuted or polysubsriwated by any of the aubstitutents described hereinabove. In another embodiment, the A ring is a tnemberec carbocyclio ring containing one or more double bonds, which ring may be unsubstitute4 monosubstittd or polysubsjituted by any of the substitutents described bmeehabove.

1

VO

0 0 fooD49) In another embodiment, the A rindg includes any type of sarataed, unsaturated or c aromatic heterocyclic ring. In another embodiment, the A ring is a 6 membered saturated heterocyclic ring, which may be unsubstiited, monosubstituted or s polysubstituted by any of the substituents described herinabove. In another 00 M embodiment, the A ring is a 5 membered saturated heterocyclic ring, which may be unsubstituted, monosubstituted or polysubstituted by any of the substitents described 0 hereinabove. In another embodiment, the A ring is a 6 membered hbeterocyclic ring Scontaining one or more double bonds, which ring may be unsubstituted, monosubstituted o io or polysubstituted by any of the substitutentsa described hereinabove. In another embodiment, the A ring is a 5 membered heterocyclic ring containing one or more double bonds, which ring may be unsubstituted, monosubstituted or polysubstituted by any of the substitutents described hereinabove. In another embodiment, the A ring is a 6 memberd hoteroaromatic ring which may be unsubstituted, monosubstituted or is polysubstitutcd by any of the substituterts described hereinabove. In another embodiment, the A ring is a 5 membered heteroaromatic ring which may be unsubstituted, monasubstituted or polysubstituted by any of the substitutents described hereinabove.

Similarly, the B ring includes any type of saturated or unsaturated carbocyclic ring. In one embodiment, the B ring is a 6 membored saturated carbocyclic ring, which may be unsubstituted, monosubstituted or polysubstituted by any of the substitutents described hereinabove. In one embodiment, the B ring is 4 5 membered saturated carbocyclic ring, which may be unsubstituted. monosubstituted or polysubstituted by any of the substitutents described hereinabove. In another embodiment, the B ring Is a 6 membered carbocyclio ring containing one or more double bonds, which ring may be unsubstituted, monosubstitued or polysubstituted by any of the substitutents described hereinabove. In another embodiment, the B ring is a 5 membered carbocyclic ring containing one or more double bonds, which ring may be unsubstituted, monosubatituted or polysubstituted by any of the substitutents described hereinabove.

In another eMbodiment, t S ring includes any type of saturted. unsaturated or aoatic heterocyctic ring. In another embodiment, the B ring is a 6 membered c-I saturted hetocyclic ring, which may be unsubstitute&, monosubstituted or polysubstituted by any of the subsdmtents described hereinabove. Int another S UmbOdiWmt the B ring is a 5 membered saturted 1 eterooyclic ring, which may be 00 Mn uusbstituted, monasubstituted or potysubstitutad by any of the SVbBtitOODIs described hereinabove. In another embodinmt the B ring is a 6 merbered hoterocyclie ring 0 containg one or more double bonds, which ring may be unsubstitutod. inonosiabstituted or polysubstituted by any of thc substitutants described hereinabove. In another o 10 embodiment, the B ring Is a 5 memabered heterocyclic ring cotaining one or more double bonds, which ring may be unsubstitutd, monosubstitutnd or polymibstted by any of the substitutonts described hereinabove. In another embodiment, the B ring is a 6 membered beteroamatic sing which may be wisubstituted, inonosubstitate:d or plysubstituted by any of the Bubstitutents described hereinabove. In another Is emibodiment, the B ring is a 5 membered heteroaromatic ring which may be unsubstituted. monosubstitutad or polys'ubstituted by any of the substitutents described hereinabove.

Nonlimiting examples of suitable A rings and/or B rings are carbocyclic, rings such as ZO cYclopontze, cyclopenten, cyckiwie 1 a=d cyclobexiene rings, and hetarocyclic rings such as pyran. dbhydropyran ten-hydropyran, pynrole, dihydropyrrole, tetrahydropyrrole, pyflzWC,. dihydropyraine, tetraydropymazlne, pyrimidine, diydropyrimidine, temraydropyrimi done, pyrazol, dihydropyrazol, tetrehydropyrazol.

piperidine, pipemaz4nc. pyridine. dihydropyridine, tem&aydtroyidine, ruorpholin; thtoruorpholine, foreAz diihydrofiam, ttrulydrofura, thiophene, dihydrathiopheno, telrahydrothlophene, dhiaole, imidazole, inoxazole, and the like.

As used herein, receptors for extracllula signaling molecules are collectively referred to as "cell signaling receptors". Many call signaling receptors cr0 tmanieinbrano proteins on a call surface; when they bind an extracellular signaling molecule a ligaa4) they become activated so as to generate a cascade of Intraocllular signals that alter the behtavior of the call. In contrast in some eases. the 1

O

O

Sreceptors are inside the cell and the signaling ligand has to enter the cell to. activate them; these signaling moleoules therefore must ie sufficiently small and hydrophobic to Cl diffe across the plasma memibane of the cell. As used herein, these receptors are collectively referred to as "intracellular cell signaling receptors", 00 C Steroid hormones are one example of small hydrophobic molecules that tt dififse directly across or are transported across the plasma membrane of target cells and 0 CN bind to intracellular cell signaling receptors. These receptors are structurally related and o constitute the intracellular receptor superfamily (or steroid-hormone receptor superfamily). Steroid hormone receptors include progesterone receptors, estrogen receptors, androgen receptors, gluoocorticoid receptors, and mineraloortlcoid,, and numerous ophan receptors. The present invention is particularly directed to androgen receptors and all of its isoforms.

In addition to ligand binding to the receptors, the receptors can be blocked to prevent ligand binding. When a substance binds to a receptor, the thrc-dimensional structure of the substance fits into a space created by the three-dimensional structure of the receptor in a ball sad socket configuration, The better the ball fits into the socket, the more tightly it is held. This phenomenon is called affinity. If the affinity of a substance is sufficiently high, it will compete with the hormone and bind the binding site more frequently. The binding of the ligand may also lead to tissue-selective recruitment of other important proteins to transduce the signal. These proteins are known as coaotivatrs and oorepressor, participate in signal tranduction, and may be selectively induced or inhibited by ligand binding. Once bound, signals may be sent through the receptor into the cells, causing the cell to respond in some fashion. This is called activation. On activation, the activated receptor then directly regulates the transcription of specific genes. But the substance and the receptor may have certain attributes, other than affinity, that activate the cell.

Chemical bonds between atoms of the substance and the atoms of the receptors may form In some cases, this leads to a change in the configuration of the receptor, which is enough to begin the activation process (called signal tansdution). As a reslt,

ID

substances can be made which bind receptors and activate them (called receptor agonists) or inactivate them (called receptor antagonists), c- The present invention is directed to selective androgen receptor modulator s compounds which are agonist compounds, and are, therefore, useful in binding to and 00 activating steroidal hormone receptors. The compounds are non-steroidal. Preferably, the _f agonist compound of the present invention is an agonist that binds the androgen o receptor. Preferably, the compound has high affinity for the androgen receptor. The IND compound may bind either reversibly or ineversibly to the androgen receptor. The o0 0 compound of the present invention may contain a functional group (affinity label) that allows alkylation of the androgen receptor covalent bond formation). Thus, in this case, the compound binds irreversibly to the receptor and, accordingly, cannot be displaced by a steroid, such as the endogenous ligands dihydrotestosterone and testosterone. It is preferable, however, for the compounds of the present invention to reversibly bind the androgen receptor.

According to one aspect of the present invention, a method is provided for binding the selective androgen receptor modulator compounds of the present invention to an androgen receptor by conacting the receptor with a selective androgen receptor modulator compound under conditions effective to cause the selective androgen receptor modulator compound to bind the. androgen receptor. The binding of the selective androgen receptor modulator compounds to the androgen receptor enables the compounds of the present invention to be useful in males and in females in a number of hormone therapies. The agonist compounds bind to and activate the androgen receptor.

Binding of the agonist compound is either reversible or irreversible, preferably reversible.

According to one aspect of the present invention, a method is provided for modulating spermatogenesis by contacting an androgen receptor of a patient with a selective androgen receptor modulator compound under conditions effective to bind the selective androgen receptor modulator compound to the androgon reoeptor and increase or decrease sperm production.

VO

0 0 According to another aspect of the present invention, a method is provided for hornonal therapy in a patient suffering from an androgen- dependent condition) which includes contacting an androgen receptor of a patient with a selective androgen receptor modulator compound under conditions effective to bind the selective androgen 00 Cc receptor modulator compound to the androgen receptor and effect a change in an tf androgn-depandent condition. Androgen-dependent conditions that may be treated 0 according to the present invention include those conditions associated with aging, such IN as hypogonadism, sarcopenia, erythopoiesis, osteoporosis, and any other conditions O 10 later determined to be dependent upon low androgen testosterone) levels. In one embodiment, the selective androgen receptor modulator compound is administered alone. In another embodiment, the selective androgen receptor modulator compound is administered in combination with progestin. In yet another embodiment, the selective androgen receptor modulator compound is administered in combination with estrogen,

JS

According to another aspect of the present invention, a method is provided for treating a subject having prostate cancer. The method comprises administrating to a subject an effective amount of a selective androgen rceptor modulator compound. In one embodiment, the selective androgen receptor modulator compound is selective for androgen or testosterone receptor.

The present invention also relates to a method of treating a subject having a chronic muscle wasting disease state which comprises contacting an androgen receptor of said subject with a selective androgen receptor modulator compound under conditions effective to bind the selective androgen receptor modulator compound to the androgen receptor and effect a change ia an androgen-dependent condition. In one embodiment, the selective androgen receptor modulator compound is selective for androgen or testosterone receptor. The present invention also relates to a method of oral administradon of the selective androgen receptor modulator compound. In one embodiment the compound is Compound I. In another embodiment the compound is Compound II. In another embodiment the compound is Compound Il. In another embodiment the compound is Compound IV. In another embodiment the compound is I I

VO

0 0 SCompound V. In another embodiment the compound is Compound VI. In another embodiment the compound is Compound VII. In another embodiment the compound is C' Compound VII.

00 cn According to one aspect of the present invention, a method is provided for binding the non-steroidal agonist compounds of the present invention to an androgen C receptor by contacting the receptor with a non-steroidal agonist compound under Sconditions effective to cause the nou-steroidal agonist compound to bind the androgen o 10 receptor. The binding of the non-steroidal agonist compounds to the androgen receptor enables the compounds of the present invention to be tseful in males and in females in a number of hormone therapies. The agonist compounds bind to and activate the androgen receptor. Binding of the agonist compound is either reversible or irreversible, preferably reversible.

According to one aspect of the present invention, a method is provided for modulating spermatogenesis by contacting an androgen receptor of a patient with a nonsteroidal agonist compound under conditions effective to bind the selective androgen receptor modulator compound to the androgen receptor and increase or decrease sperm production.

According to another aspect ofthe present invention, a method is provided for hormonal therapy in a patient one suffering from an androgen- dependent condition) which includes contacting an androgen receptor of a patient with a nonsteroidal agonist compound under conditions effective to bind the non-staroidal agonist compound to the androgen receptor and efftt a change in an androgen-dependent condition. Androgen-dependent conditions that may be treated according to the present invention include those conditions associated with aging, such as hypogonadism, sarcopenia, erythropoiesis. osteoporosis, laok of sexual libido and any other conditions later determined to be dependent upon low androgen testosterone) levels. In one embodiment, the non-steroidal agonist compound is administered alone. In another embodiment, the non-steroldal agonist compound is administered In combination with I I

VO

0 progestin. In yet another embodiment the non-sterodal agonist compound is administered in combination with estrogen.

According to another aspect of the present invention, a method is provided for S treating a subject having prostate cancer. The method comprises administrating to a 00 subject an effective amount of a non-steroidal agonist compound. In one embodiment kn the non-steroidal agonist compound is selective for androgen or teastosterone receptor.

INDThe compounds ofthe present invention have an assymetric center and can be the R or S isomer, or a mixture of both. In one embodiment, the compounds raotmic mixaes of the R and S enantiomers. In another embodiment, the compourands are substantialy pure i onaniomers. In another embodiment the compounds are subsantially pure S nantiomers. "Substantially pure" is defined herein as greater than about 95% preponderance of one somer Where the above-described processes for the is preparatIon of the compounds of use in the invention give rise to mixtures of stercoisomers, these isomers may be separated by conventional techniques, such as preparative chromatography. The compounds may be prepared in racemic farm, or individual enantiomners may be prepared either by anantiospecific synthesis or by resolution As used hcmin "pharmsaceutical composition" moans therapeutically effective amountS of the SARM or the non-stearoidal agonist compound of the present invention, together with suitable diluenta, preservatives, solubilizers emulsifiers, ajnvant and/or carriers. A "therapeutically effeotive amount" as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen. Such compositions are liquids or Lyophilized or otherwise dried formulations and nclude diluents of various buffer content Tris-HCI.. acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents Tween 20, Tween 80, Pluronic P68, bile acid salts), solubllizing agents 3o glycerol, polyethylene glycerol), anti-oxidants aooatbic acid, sodiug metabisulfite), preservatives Thimerosal, bonzyl alcohol, parabens), bulking substances or tonicity modifiers lactose, mannital), covalent attachment of

VO

0 polymers such as polyethylene glycol to the protein, complexation with metal ions, or Iacorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolio acid, hydrogels, etc, or onto liposomes, microemulsions, micalles, unilamellar or multilanmellar vesicles, erythrooyte ghosts, or s5 spharoplasts. Such compositions will influence the physical state, solubilty, stability,

O

en rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots fatty acids, waxes, oils).

0 o Also comprehended by the invention are particulate compositions coated with o o polymers poloxamers or poloxamines). Other embodiments of the compositions of the invention incorporate particulate forms, protective coatings, protesse inhibitors or permeation arenhancers for various routes of administration, including parenteral, pulmonary, nasal and oraL In one embodiment the pharmaccutical composition is administred parenterally, pamoancerally, transmucosally, tansdermally, Is intramuscularly, intravenously, lntradermaUy subcutaneously, intraperitonealy, intraventrialsrly, intracranially and intratumorally.

Further, as used herein '"pharmnaceutically acceptable carriers" are well known to those skilled in the art and include, but ane not limited to, 0.01-0.1M and pretbrably O.OSM phosphate buffer or 0.8% saline. Additionally, such phannaceutically acceptable caniers may be aqueous or non-aqueous solutions, suspensions, and emulsions.

Examples of non-aqueous solvents am propylene glycol, polythylene glycol, vegetable oils suchb as olive oil. and injootable organic esters such as ethyl oleate. Aqueous caniers include water, alcoholicaqueous solutions, emulsions or suspensions, including saline and buffered media.

Parenteral vehicles include sodium chloride solution. Ringer's dextross, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replanrishers, electrolyte replenishers such as those based on inger's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobils, antioxidants, collating agents, inert gases and the like.

VO

0 0 CL| Controlled or sustained release compositions include fonnulation in lipophilic depots fany acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers poloxamers or poloxamines) and the compound coupled to antibodies directed against tissuespecific receptors, lgands or antigens or 00 coupled to igands oftissue-speific receptors.

c, t r SOther embodiments of the compositions of the invention incorporate particulate forms, protective coatings, protease inhibitors or permeation enhancers for S1to various rutes of administration, including paroeteral, pulmonary, nasal and oral.

Compounds modified by the covalent attacment of water-soluble polymers such as polyethylene glycol, oopolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dectran. polyvinyl alcohol, polyvinylpyrrolidone or polyproline am known to exhibit substantially longer half- lives in blood following intravenous injection than do the corresponding unmodified compounds (Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987). Such modifications may also increase the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound. and greatly reduce the immunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity may be achieved by the administration of such polymer-compound abducts less frequently or in lower doses than with the unmodified compound.

In yet another embodiment, the pharmaceutical composition can be delivered in a controlled release system. For example, the agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposornes, or other modes of administration. In one embodiment, a pump may be used (see Langer.

supra; Sefton, CRC Crit. ReE Biomed. Eng. 14:201 (1987); Buobwald at al.. Surgery 88:507 (1980); Saudek at al., N. Bgl. J. Med. 321:574 (1989). tI another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity to the therapeutio target, the brain, thus requiring only a friation of the systemic dose (sec, Ooodson, in Medical Applications of Controlled

VO

0 SRelease, sopa, voL 2, pp. 115-138 (1984). Preferably, a controlled release device is introduced into a subject in proximity to the site of inappropriate immune activation or a nmnor. Other controlled release systems are discussed in the review by Leager (Science 249:1527-1533 (1990).

00 The pharmaceutical preparation can comprise the selective androgen receptor tr modulator alone, or can further include a pharmaceutically acceptable carrier, and can be in solid or liquid form such as tablets, powders, capsules, pellets, solutions, suspensions.

Clixirs, emulsions, gels, creams, or suppositories, including rectal and urethral io suppositories. Pharmaceutically acceptable carriers include gams, starches, sugas, cellulosic materials, and mixtures thereof. The pharmaceutical preparation containing the selective androgea receptor modulator can be administered to a subject by, for example, subcutaneous implantation of a pellet; in a further embodimeat, the pellet provides for controlled release of selective androgen receptor modulator over a period of time. The preparation can also be administered by intravenous, intrarterial, or intramuscular injection of a liquid preparation, oral administration of a liquid or solid preparation, or by topical application. Administration can also be accomplished by use of a rectal suppository or a urethral suppository.

The pharmaceutical preparations of the invention can be prepared by known dissolving, mixing, granlatig, or tablet-foring processes. For oral administration, the selective androgen receptor modulators or their physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into a suitable form for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions. Examples of suitable inert vehicles are conventional tablet bases such as lactose, sucrose, or ornstarch in combination with binders like acacia, cornstarch, gelatin or with disintegrating agents such as cornstarch, potato starch, algino acid, or with a lubricant like steario acid or magnesium stearate.

Examples of suitable oily vehicles or solvents are vegetable or animal oils

VO

0 0 c such as sunflower oil or fish-liver oil. Preparations can be effected both as dry and as wet granules. For parenteMal administration (suboutaneous, intravenous, intraarterial, or intramuscular injection), the SARM agents or the non-steroidal agonist agents or their physiologically tolerated derivatives such as salts, eaters, N-oxides, and the like arc s converted into a solution, suspension, or emulsion, if desired with the substances 00 customary and suitable for this purpose, for example, solubilizers or other auxiliaries.

In Examples are: sterile liquids such as water and oils, with or without the addition of a o surfactant and other pharmacetically acceptable adjuvants. Illustrative oils arc those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextros and related sugar solutions, and C glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

The preparation of pharmaceutical compositions which contain an active 1i component is well understood in the'art Typically, such compositions are prepared as aerosols of the polypoptide delivered to the nasopharynx or as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapautic inrediant is often mixed with exciplents that are pharmaceuticatly aoceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.

In addition, if desired, the composition can contain minor amounts of auxiliary 2 substances such as wetting or emulsifying agents, pH buffering agents, which enhance the effectiveness of the active ingredient An active component can be f mnlated into the composition as neutralized pharmaceutically acceptable salt forms. Pharmaoeically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule), which arc formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic aoids as acetic, Oxalio, tartaria, mandelic, and the like.

I I O IND 0 SSalts formed from the fme carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium ammonium, calcium, or ferri hydroxides, and such organic bases as isopropylamine, trimethylaminc, 2-ethylamino ethanol, histidino, procaine, and the liue.

00 Mn For topical administration to body surfaces using, for example, creams, gels.

drops, and the like, the SARM agents or the non-agonist steroidal compounds or their o physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are prepared and applied as solutions, suspensions, or emulsions in a physiologically 0 10 acceptable diluent with or without a pharmaceutical carrier.

in another embodiment the active compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat at al., in Liposomes in the Therapy of nfectious Disease and Cancer, Lopez- Berestein and Fidler is Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).

For use in medicine, the salts of the SARM or the non-steroidal agonist compounds will be phanaceutioally acceptable salts. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acoptable salts of the compounds of this invention include acid addition salts, which may, for example, be formed by mixing a solution of the compound according to the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid. sulphuric acid mothanesulphonic acid, fAmario acid, malcic acid, succinic acid, acetic acid, benzoic acid, oxalic said, citic acid, trtario acid, carbonic acid or phosphoric acid.

The present invention further relates to a method of determining the presence of a selective androgen modulator compound and/or a non-steroidal agonist compound of the present invention in a sample. The method comprises the steps of obtaining the sample, and detecting the compound in the sample, thereby determining the presence of the oompound in the sample.

I

O

0

O

In one embodiment, the sample is a blood serum sample. In another Sembodiment, the sample is a plasma sample. In another embodiment, the sample is a urine sample. In another embodiment, the sample is a saliva sample. In another s embodiment, the sample is any other tissue sample.

00 In In one embodiment, the detection step comprises measuring the absorbance of 0 the compound at a predetennined wavelength. For example, the compounds of the 0D present invention absorb in the ultraviolet region of the spectrum, with an absorbency peak at 270 am. Thus, in one embodiment of the present invention, the compound is detected by monitoring the UV absorbance of the sample at 270 nm. It should be noted that the present invention is not limited to UV absorption, and that any other spectrometric methods of identification are applicable. For example, compounds can be detected by measuring their infra-red or visible absorbance.

Is In another embodiment, the present invention further provides a method of dotenriniog the concentration of a selective androgen receptor modulator compound and/or a non-steroidal agonist compound of the present invention in a sample. The method comprises the steps of obtaining a sample: determining the level of the compound in the sample, and calculating the concentration of the compound in the sample by comparing the level with a standad sample containing a known concentration of the compound. Calibration curves of known concentrations of the compound in the sample, can be obtained, and the concentration of the compound in the test sample is calculated therefrom. By "level" it is meant te absorption level of the compound at the measured wavelength.

In another embodiment, the compound is detected in the sample by contacting the sample with a binding protein which specifically binds to the compound, and determiing the amount of binding protein bound to the compoimd, The concentration of the compound can be determined by measuring the amount of binding protein bound to the compound, and comparing that amount to a standard sample containing a known conontration of the compound binding protein complex.

VO

0 0 O Protein levels can be deteonined according to standard techniques, as described in Sambrook et al. Briefly, a sample obtained from a subject is contacted with a binding protein which specifically binds to a specific compound of the present s invention, and the amount of complex fonned between the binding protein and the 00 compound is detennined. In one embodiment, the binding protein is an antibody which in specifically binds to one or more compounds of the present invention. In another o embodiment, the binding protein has a detectable label bound thereto, and the complex Sbetween the binding protein-label compound is determined by visualizing the complex.

o C As defined herein. "contacting" means that the binding protein is introduced into the sample in a test tube, flask, tissue cutue, chip, array, plate, microplate, capillary, or the like, and Incubated at a temperature and time sufficient to permit the binding component to bind to a cell or a fraction thereof or plasma/serum or a fraction s1 thereof containing the target. Methods for contacting the samples with the binding proteins, or other specific binding components are know to those skilled in the art and may be selected depending on the type of assay protocol'to be run. Incubation methods art also standard nd are known to those skilled in the art.

"Visualizing" the complex may be carried out by any means known in the ar, including, but not limited to, ELISA, radioimmunoassay, flow cytometry, dot blots, western immunoblotting combined with gol eletrophoresis, imrunohistochenistry at light and electron pe levels, HPLC and mass spectromctry.

Bither monoolonal or polyclonal antibodies (as well as any recombinat antibodies) specific for the selective androgen modulator compounds or the nonsteroidal agonist compounds of the present invention can be used in the various immunoassays. The antibodies may be detootably labeled, utilizing conventional labeling techniques well-known to the art. As used herein, the term "label" refers to a molecule, which may be conjugated or otherwise attached covalently or noncovalently) to a binding protein as defined herein. Labels are known to those skilled in th art. Thus, the antitodies may be labeled with radioactive isotopes, on-radioactive i

VO

0 0 isotopic labels. fluorescent labels, enzyme labels, chemiluminescant labels, biobmincemt labels, free radical labels, or bacteriophage labels, using techniques known in the art. Examples of radioisotopic labels are .sup.3 I, .sup 1 25 I, .sup.131 1, .sup. 14 C, etc. Examples of non-radioactive isotopic labels are .sup.55 Mn, s .sup.56 Fe, etc. Examples of fluorescence labels are fluorescent labels which are directly labeled with the preferred fluorescence label, or fluorescent labels which are indirectly labeled with the preferred fluorescence label. In the last case, the preferred fluorescence label is conjugated to a secondary antibody, which is directed against the first antibody, such as an anti species Ig antibody. Typical fluorescent labels include, but N 10 are not limited to a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, etc., for example fluorescein isothiocyanate (FITC, International Biological Supplies. Melbourne, FL), rhodamine, phyooerythrin Coulter Corp., HM415a, phycocyanin, alophycocyanin, phycoerythrin-cyanin dye 5 Coulter), label, a phycocyanin label, an allophycocyanin label, an O-phthaldehyde label, IS a fluarescamine and Texas Red.

Examples of enzyme labels include alkaline phosphatase, beta-galactosidase, glucose-6-phosphate dehydrogenase, maleate dehydrogenase, and peroxidase. Two principal types of enzyme immunoassay are the enzyme-linked immunlmosorbent assay (ELISA), and the homogeneous enzyme immunoassay, also known as enzyme-multiplied imrnnoassay (EMIT, Syva Corporation, Palo Alto, CA).

In the ELISA system, separation may be achieved, for example, by the use of antibodies coupled to a solid phase. The EMIT system depends on deactivation of the enzyme in the tracer-antibody complex; the activity can thus be measured without the need for a separation step.

Particularly suimtable labels include those, which permit analysis by flow cytometry, fluorochromes. Other suitable detectable labels include those useful in colorimetric enzyme systems, e. horseradish peroxidase (HRP) and alkaline phosphatase Other proximal enzyme systems are known to those of skill in the art, including hexokinase in conjunction with glucose-6-phosphate dehydrogenase.

1

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0 Additionally, chemiluminensoent compounds may be used as labels.

Chcmiluminescent labels, suchb as green fluorescent proteins, blue fluorescent proteins, sad variants thereof are known. Also bioluminescence or ohendtilumrninescence can be detected using& respectively, NAD oxidoreductase with luciferase and substrates NADb s and FNIN or peroxidaseowith luminol and substrate peroxide. Typical chmiluminescent 00 compounds include luminol, isoluminol, aromatic aoridinium caters, imidazoles, In acridinium salts, and oxalate esters. Similarly, bioluminescent compounds may be o utilized bfor labelling, the bioluminescent compounds including lociferin, luciferase, and Sacquorin. Once labeled, the antibody may be employed to identify and quantWify o to immunologic counteparts (antibody or anrigenic polypeptide) utilizing toohniques well-known to the art The following examples are presented in order to more fully illustrate the is preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXPERIMNTAL DETAILS SEC ON EXSAMPE 1 Nonsteroidal Lignds with Androgenic and Anabolic Activity The SARM compounds provided herein were designed, synthesized and evaluated for in-vitro and in-viva pharmacologic activity. The in-vitro androgen receptor binding affinity and ability to maintain androgen dependent tissue growth in oastrated animals was studied Androgenic activity was monitored as the ability of the SARM compounds to maintain and/or stimulate the growth of the prostate and seminal vesicles as measured by weight. Anabolic activity was monitored as the ability of the SARM compounds to maintain and/or stimulate the growth of the levator ani muscle, as measured by weight

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0 c Synthefic Procdures f CoPopouud I-VIIIt (2R)--Metbacryloylpyrrodi-2-carboxyic Acid (R-129). D-Proline (R- 128, 14.93 g, 0.13 mol) was dissolved in 71 nmL of 2 N NaOH and cooled in an ice bath; the resulting alkaline solution was diluted with acetone (71 mL). An acetone solution (71 00 mL) of motacryloly chloride 127 (13.56 g, 0.13 mol) and 2N NaOH solution (71 mL) M were simultaneously added over 40 min to the aqueous solution of D-proline in an ice tn bath. The pH of the mixture was kept at 10-ll"C during the addition of the mnetacryloly 0 S. chloride. After stirning (3 h, room temperarre), the mixture was evaporated in vacuo at a temperature at 35-45 "C to remove acetone. The resulting solution was washed with ethyl ether and was acidified to pH 2 with concentrated HC. The acidic mixture was saturated with NaC and was extracted with EtOAc (100 mL x The combined extracts were dried over NaS04, filtered through Celite, and evaporated in vacuo to give the crude product as a colorless oil. Recrystalliation of the oil from ethyl other and bhxanes is afforded 16.2 of the desired compound as colorless crystals: mp 102-103 0 C (lit [214] mp 102.5-103.5 the NMR spectrum of this compound demonstrated the existence of two rotamers of the title compound. 'H NMR (300 MHz. DMSO-d) 6 5.28 and 5.15 for the first rotamor, 5.15 and 5.03 for the second rotamer (totally 2H for both rotamers, vinyl CH2), 4.484.44 for the first rotamer, 4.244.20 for the second rotamer (totally 1I for both rotamers, CH atthe chiral center), 3,57-338 2H, CH2), 2.27-2.12 (18, CH), 1.97-1.72 6H, CH2,'CH, Mo); 3 C NMR (75 MHz, DMSO-d) 8 for major rotamer 173.3. 169.1, 140.9, 116.4, 58.3. 48.7, 28.9, 24.7, 19.5: for minor rotamner 174.0, 170.0, 141.6, 115.2, 60.3, 45.9, 31.0, 22.3, 19.7; IR (KBr) 3437 1737 1647 (CO, COOH), 1584, 1508, 1459, 1369, 1348, 1178 omr; +80.80 (c 1, McOH); Anl. Calod. for C 59.00, R 7.15, N 7.65.

Found: C 59.13, H 7.19, N 7.61.

(3R,8aR)-3-Bromomethyl-3-met h y ydro-pyrralo[2,1c)[l,4laxazine-,4-dlone R-130). A solution of NDS (23-.5g, 0.132 mol) in 100 mL of DMF was added dropwise to a stirred solution of compound R-129 (16.1g, 88 rmmol) in 70 mL of DMP under argon at room tmnporatlre, and the resulting mixture 9was stirred 3 days. The solvent was removed in vacuo, and a yellow solid was precipitated.

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0 The solid was suspended in water, stirred overnight at rmom temperature, filtered, and dried to give 18.6 (smaller weight when dried 34%) of the title compound as a yellow solid: mp 152-154 'C (lit. [214] mp 107-109 OC for the S-isomer); 'H NMR (300 MHZ. DMSO-d) 8 4.69 (dd, J 9.6 Hz, J 6.7 Hz, I, CH at the chiral center), 4.02 J- 11.4 Hz, 1, CHH.), 3.86 J- 11.4Hz, IHCHb), 3.53-3.24 4H, CH).

00 2.30-2.20 IH, CH 2.04-1.72 3H, CH and CH), 1.56 2, Mc); '3C NMR MHz, DMSO-s) 6167.3, 163.1, 83.9, 57.2, 45.4, 37.8, 29.0,22.9, 21.6; IR Kar) 3474, 1745 1687 1448. 1377, 1360, 1308, 1227, 1159, 1062cmr'; [a]26+124.5 IND (c 1.3, chloroform); Anal. Caled. for Cs, 1 2 BrN0 3 C 41.24, H 4.61, N 5.34. Found: o 10 C 41.46, H 4.64, N 5.32.

(2R)-3-Bromo-2-bydroxy-2-methylprepanolC Acid (R-131). A mixture of bromolactonc R-130 (18.5g 71 mmol) in 300 mL of 24% HBr was hated at reflux for I b. The resulting solution was diluted with brine (200 mL), and was extracted with ethyl is acetate (100 mL x The combined extracts were washed with saturated NaHCO 3 (100 mL x The aqueous solution was acidified with concentrated HCI to pH 1, which, in turn, was extracted with ethyl acetate (100 mL x The combined organic solution was dried over Na 2 SO4, filtered through Celite, and evaporated in vauuo to dryness.

Recystallization from toluene afforded 10.2 g of the desired compound as colorless orystals: mp 107-109 9C (Ut. [214) mp 109-113 "C for the S-isomrner); 'R NUR (300 MHz, DMSO-d) 6 3.63 (d J 10.1 Hz, LH, CHH.), 3.52 J 10.1 Hz, 1L, CHH), 1.35 (s,3H, Me); IR (KBr) 3434 3300-2500 (COOH), 1730 1449, 1421, 1380, 1292, 1193, 1085 cm'; la.i +1O0.5" (c 2.6, MeOH); Anal. Calcd. for C4HBzO: C 26.25. 8 3.86. Found: C 26.28, H 3.75.

N-(4-Nitro-3-(trfluoroethyOphnyll-(2R)-3-brom 2-hydroxy-2methylprepanamlde (R-132). Thionyl chloride (8.6 g, 72 mmol) was added dropwise under argon to a solution of bromoacid R131 (11.0g. o60 unol) in 70 mL of DMA at to -10 The resulting mixture was stirred for 2 h under the same conditions. A solution of4-itro-3-triflucromethyl-aniline (12.4 60 mmol) in 80 mL of DMA was added dropwise to the above solution, and the resulting mixture was stirred overnight at toom temperatum. The solvent was removed on Rotavapor using high vacuum oil pump;

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0 the residue was dilated with aturated NaHCOh solution, and extracted with ethyl other (100 mL x COmbined extracts were dried over anbydrous N 2 S0 4 filtered through Colite, and purified by flash chromatography on silica gel, using methylene chloride as eluent to afford 18.0 g of the desired compound: mp 98-100 'C (R 0.2 silica gel, CHC1 2 'H NIR (300 MHWz, DMSO-d) 8 10.54 1, 8.54 J 2.1 Hz, 00 1H, ArH), 8.34 (dd, J 9.0 11H, J 2.1 Hz. 1, ArH), 8.18 J 9.0 Hz, 1H. ArH), 6.37 1W, OH), 3.82 J 10.4 RHz, 1H CHHC), 3.58 3 10.4 Hz. 1H, CM), 0~1.48 31, Mc); 'C NMR (75 MHz, DMSO-d) 8 173.6 (C=1OX 143.0, 127.2, 123.2, 122.6 (qc, J 33.0 Hz) 122.0 J -271.5 Hz), 118.3 J 6.0 HZ), 74.4, 41.4, 24.9; IR (KBr) 3344 1680 1599, 1548 Ar), 1427. 1363, 1161 om'; MS (ESI): m/z 370.8 Anal. Caled. for CuIHioBrN 2 04: C 35.60, H 2.72, N 7.55. Found: C 35.68, H 2.72, N 7.49.

N-14-lntro-3-trifloromethyolphenyll-(25)-3-14-(actlmno)phenoxy]-2hydroxy-2-mthylpropanamide (8-147). The title compound was prepared from compound R-132 (0.37 g, 1.0 mmol), 4-actamidphenol (0,23 g. 1.5 mmol) K) 2 C0 3 (0.28 g, 2.0 mml). and 10% of benzylrlbutylammonium chloride as a phase transfer catalyst In 20 mnL of methyl ethyl ketone was heated at reflux overnight under argon.

The reaction was followed by TLC, the resulting mixture was filterd through Celite,.

and concentrated in vacuo to dryness. Purification by flash column chromatography on silica gel (hxanes-ethyl acetate, 3:1) yielded 0.38 g (Ri 0.18 hxanea-thyl acetate, 3:1) of the desired compound as a light yellow powder: mp 70-74 1C; The solid can be recrystalized from ethyl acetate and hexane); 'H NMR (300 MHz, DMSO-de) b 10.62 11, NH), 9.75 1K NI), 8.56 J 1.9 Hz, 1H, ArH), 8.36 (dd, J= 9.1 z, 1 1.9 Hz, 1i. ArH), 8.18 9.1 Hz 1H, ArH), 7.45-7.42 (nm, 21, ArN), 6.85-6.82 2H, Ar). 6.25 11, OH), 4.17 (4 J 9.5 Hz, 1, CHi), 3.94 J 9.5 Hz, !H, CHhb). 1.98 3H, Me), 1.43 3W, Mo); 3 C NMR (75 MHz, DMSO-ds) 8 174.6 167.7, 154.2, 143.3, 141.6, 132.8, 127.4, 123.0, 122.7 J= 33.0 Hz), 122.1 (q, J 271.5 Hz), 120,1, 118.3 I 6.0 Ha), 114.6, 74.9, 73.8, 23.8, 23.0; IR (KBr) 3364 (OH) 1668 1599, 1512 Ar)6 1457, 1415, 1351. 1323, 1239, 1150 1046 cm MS minz 464.1 Anal. Calcd. for CloHsPNsO.: C 51.71, H 4.11, N 9.52. Found: C 52.33, H 4.40, N 9.01.

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c en 0 cO IN The in-vitro activity of the SARM compounds, specifically compound VII, 0 demonstrated high androgen receptor binding affinity (Ki 7.5 nM). Animal studies with the SARM compounds, specificay compound V, demonstrated thai it is a potent androgenia and anabolico nonsteroidal agent Four groups of rats were used for these studies: intact controls, castrated controls, astrated animals neated with teestosterone propionate (100 pg/day), and castrated animals treated with compound V (1000 4day). Testosterone and compound VII were delivered at a constant rate for 14 days via subcUtaneous osmotic pumps.

The results of these studies are shown in Figure 1. Castration significantly reduced the weight of androgenic prostate and seminal vosicles) and anabolic levaror ani muscle) tissues, but had lirde effect on animal body weight Treatment of castrated animals with testosterone propionte or compound VII maintained the weight of androgenic tissues to the same degree. Compound VII had similar androgenic activity as testosterone propionate the prostate and seminal vesicle weights were the same), but much greater efficacy as an anabolic agent. Compound VII showed greater anabolic activity than testosterone propionate at the doses tested the levator ani muscle maintained the same weight as intact control animals and was greater than that observed for testosterone). The experiments presented herein are the first in-vivo results which demonstrate tissue-selective androgenic and anabolic activity differing androgenia and anabolic potency) of a nonstoroidal ligand for the androgen receptor.

Va 0 0, EXAMPLE 2 Nonsterodal Ligands with Androgenic and Anaboli Activity The in-vive efficacy and acute toxicity of four novel nonsteroidal androgens (compounds IV, V. V1 and VII) in rats was examined. In-vitro assays established that these compounds bind the androgen receptor with very high affinity. The structures and names of the four compounds are presented below: GTx-l 14 OTx-015 GTx-016 GTx-017 R- F R**COCH3

R-OC

2 11 R-NHCOCH3 EXPERIMENTAL METHODS Material The S-isomers of compounds GTx-014 (compound IV), GTx-O1S (compound OTx-016 (compound and OT-007 (compound VII wherin R is NHCOCH3) and the R-isomer of GTx-014 were synthesized in accordance with the scheme as set forth in Figure 9. Testostrone propionate polyethylene glycol 300 (PE0300, reagent grade) and neutral buffered formalin (10% wtv) were puchased fom

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Sigma Chemical Company (St Louis, MO). Alzet osmotic pumps (model 2002) were Spurbased from Aza Corp. (Palo Alto, CA).

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Animals. Immature male Sprague-Dawley rats, weighing 90 to 100g, were purchased from Harlan Biosciences (Indianapolis, IN). The animals were maintained on 00 a 12-hour light-dark cycle with food and water available ad libitum..The animal protocol r was reviewed and approved by the Instituional Laboratory Animal Care and Use Committee.

Study Desgn. Rats were randomly distributed into twenty-nine (29) groups, with animals per group. Treatment groups are described in Table 1. One day prior to the start of drug treatment, animals in groups 2 through 29 were individually removed from the cage. weighed and anesthetized with an intraperitoneal dose of ketamineaxylazine (87/13 mg/kg approximately I mL per kg). When appropriately anesthetized no response to toe pinch), the animals' cars were marked for identification purposes.

Animals were then placed on a sterile pad and their abdomen and scrotum washed with betadine and 70% alcohol. The testes were removed via a midline scrotal incision, with sterile suture being used to ligate supra-tesldcular tissue prior to surgical removal of each testis. The surgical wound site was closed with sterile stainless steel wound clips, and the site cleaned with betadine. The animals were allowed to recover on a sterile pad (until able to stand) and then returned to their cage.

Twenty-four hours later, animals in groups 2 through 29 were -anesthetized with ketamine/xylazine, and an Alzet osmotic pump(s) (model 2002) was placed subcutaneouly in the scapular region. In this instance, the scapular region was shaved and cleaned (betadine and alcohol) and a small incision (1 cm) made using a sterile scalpel. The osmotic pump was inserted and the wound closed with a sterile stainless steel wound clip. Animals were allowed to recover and were returned to their cage.

Osmotic pumps contained the appropriate treatment (designated in Table 1) dissolved in polyethylene glycol 300 (PBE300). Osmotic pumps were filled with the appropriate solution one day prior to implantation. Animals were monitored daily for signs of acute toxicity to drug Uteatment lethargy, rough coat).

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0 After 14 days of draug treatment, rats were anesthetized with keamineylazine. Animals wore then pacrificed by exsanguinations under anesthesia.

A blood sample was collooected by venipuncture of the abdominal aorta, and submitted for complete blood cell analysis. A portion of the blood was placed in a separate tube, centrifuged at 12,000g for 1 minute, and the plasma layer removed and fozen at 00 SThe ventral prostates, seminal vesicles, levator ani muscle, liver, kidneys, spleen, lungs, and heart were removed, cleared of extraneous tissue, weighed, and placed in vials 0 containing 10%/ neutral buffered formalin. Preserved tissues were sent to GTx, Inc. for o bistopathological analysis.

For data analysis, the weights of all organs were normalized to body weight and analyzed for any statistical significant difference by single-factor ANOVA. The weights of prostate and seminal vesiole were used as indexes for evaluation of androgenic activity, and the levator ani muscle weight was used to evaluate the anabolic activity.

RESULTS

The androgenic and anabolic activities the S isomers of compounds GTx-014, OTx-015. GTx-016 and GTx-007, and the R isomer of GTx-014 ware examined in a castrated rat model after 14 days of administration. Testostarone propionatt, at increasing doses, was used as the positive control of anabolic and androgenic effects.

As shown in Figures 2 and 3. the weights of prostate, seminal vesicle, and levator ani musole in castrated, vebhiole-troted rats decreased significantly, due to the ablation of endogenous androgen production. Bxogcnous administration of teststerone propionate an androgenic .and anabolic steroid, increased the weights of prostate, seminal vesiclo, and levator ani muscle in castrated rats in a dose-dependent manner.

The R-isomer of GTx-014, and S-isomers of CTx-015 and OTx-016 showed no effect on the weights of prostate, seminal vesiole, and levator ani muscle in castrated animals (data not shown). The S-isomers of GTx-007 (Figure 2: S-OTx-007) and GTx-014 (Figure 3: S-GTx-014) resulted in dose-dependent increases in prostate, seminal vesicle and levator ni muscle weights. Compared with testosterone propionate, S-OTx-007

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c showed lower potency and intrinsic activity in increasing the weights of prostate and Sseminal vesicle, but a greater potency and intrinsic activity in increasing the weight of levator ani muscle. Particularly, S-GTx-007, at a dose as low as 0.3 mg/day, was able to maintain the levator ai muscle weight of castrated animals in the same level as that of intact animals. Thus, S-OTx-007 is a potent nonsteroidal anabolic agent with less 00 androgenic activity but more anabolic activity than testosterone propionate. This is a tn significant improvement over previous claims, in that this compound selectively Sstimulates muscle growth and other anabolic effects while having less effect on the ND prostate and seminal vesicles. This may be particularly relevant in aging men with o 0o concerns related to the development or progression of prostate cancer.

Tx-014 was less potent than GTx007, but showed greater tissue selectivity (compare effects on the prostate and seminal vesicles in Figures 2 and GTx-014 significantly increased levator ani muscle weights, but showed little to no ability to is stimulate prostate and seminal vesicle growth the prostate and seminal vesicle weights were less than 20% of that observed in intact animals or in animals treated with testosterone propionate), Results showed that none of the examined compounds produced significant effect on body weight or the weights of other organs liver, Iddneys, spleen, lungs and heart). Nor did any compound produce any signs of acute toxicity, as gauged by diagnostic hematology tests and visual examination of animals receiving treatments.

Importantly, OTx-007 did not suppress the production of luteinizing hormone (LH) or follicle stimulating hormone (FSH) at a dose of 0.3 mg/day a dose that exhibited maximal anabolic effects).

In summary, S-GTx-007 exhibited exceptional anabolio activity in animals by maintaining the weight of levator ani muscle after removal of endogenous androgen.

This discovery represents major progress towards the development of therapeutically useful nonsteroidal androgens, and a major improvement tissue selectivity and potency) over previous drugs in this class. S-GTx-014 and S-OTx-007 showed selective anabolic activity in comparison with tastosterone propionate, an androgenic and anabolic r

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C steroid. The tissue-selective activity is actually one of the advantages of nonsteroidal Sandrogens in terms of anabolic-rlated applications.

S Despite similarities in structure and in-vitro functional activity, the S-isomers of compounds GTx-014, OTx-015, GTx-016, and GTx-007 exhibited profbund 0 0 differences in terms of their in-vive activity. OTx-007 the most efficacious androgenic ln and anabolic activity in animals, with the anabolic activity greater than that of O testosterone propionate. GTx-014 showed a small degree of androgenic activity, but an N anabolie activity comparable to testosterone propionate. In contrast, GTx-015 and GTx- 0 o 0 016 failed to produce any androgenic or anabolic activity in-vivo.

These studies show the discovery of two members (GTx- 0 14 and OTx-007, compounds, compounds II and V respectively) of a new class of selective androgen receptor modulators (SARMS) that demonstrate potent anabolic effects muscle growth) with less androgenic activity prostatic growth). This new class of drugs has several advantages over non-selective androgens, including potential therapeutic applications in males and females for modulation of fertility, erythropoiesis, osteoporosis, sexual libido and in men with or at high risk for prostate cancer.

Further, Figures 7 and 8 demonstrate the effects of GTx-014 and OTx-007 on LI and FSH levels in rats. These results further demonstrate the novelty of these SARMs, due to their differential effects on these reproductive hormones, thus demonstrating the tissue-specific pharmacologic activity. In Figure 7, LH levels in castrated animals treated with TP and GTx-014 were significantly lower than those of untreated animals castrated controls) at doses greater than or equal to 0.3 mg/day.

However, higher doses 0.5 mg/day or higher) of GTx-007 were required before significant decreases in LH levels were observed. Thus, GTx-007 does not suppress LH levels at doses that are capable of eliciting maximal stimulation of levator ani muscle growth. In Figure 8, FSH levels in castrated animals treated with GTx-014 were significantly lower than those of untreated animals castrated controls) at doses of mg/day or higher. Similarly, lower FSH levels were observed in animals treated with TP. However, only this difference was only significant at a dose of 0.75 mg/day. PSH Va 0 0 ci ci levels in anlals treted with GTc-007 were not sipfcmtly differet frM those of anWd ajimas at any dose level tested. Thus, QTx-007 does act suppress FSU levels at doses that are capble of eliciting maximal stimulation of )avatar ani muscle growth- Table 1. Animal. Groups and £xperLznuntal Design Group Castrated? Drug T00ii of Oma~hI I Nr None None___ 3 Ye Teststerone .1 wg/diy 4 Yes Tets~n 03T ii1/iay V Testosteroe O. MM da Yes Tistiuiem 0.7 mg/day Yes eSttoe 1.0 in day 8 yes 14 Yes iZO~bIF 0.1 mg/day i1 yes SGx045- 16s F8 T§WISR 0.75 mg/day ThZi .7 g/day S Yeas_ 17 yes -GTij0ir 01 g/day 23 y V S-GTXc-O16 TYO&/d 2w Yes

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27 Ye 1S Tff 0.5 in day S 29- Yes None 1

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0 0s ci 00 E& L SEXAWIPLE 3 Pharmackldnetdes of GTx-007 In Dogs o 10 The pbamuscokinetics of S-GTx-007, a .novel selective androgen receptor o modulator (SARM), were characterizd in beagle dogs. A four-treatmn, four-period crossover design was utilized in the study, which involved a total of six beagle dogs, thrucf f each gender. Each animal received a 3 mg/kg IV dose, a 10 mg/kg TV dose, a mg/kg PO dose in solution, and a 10 mg/kg PO dose in capsule, in a randomly assigned Is order. There was an one-week washout period between treatments. Plasma samples wereo collected for up to 72 br after drug administration. Plasma S43Tx-007 concentrations were analyzed by a validated IPLC method. The clearance volume of distribution (Vas), half-life and other pharmacokinctic parameters were determined by noncomnpartmental methods. Results showed that S-GTx-007 was cleared from dog plasma with a terminal Tie of about 4 hr and a CL of 4.4 mllnintg after IV administration. Figures 4, 5, and 6 show the plasma concentration-time profiles of S- GTx-007 after administration of an intravenous solution, oral solution, and oral capsule, respectively. The pharmacokinetics were dose- and gender-independent. The oral bicavailability of S-GTx-007 varied with the dosage form, and averaged 38% and 19% for solution and capsule, roapectively. Thus, S-GTx-007 demonstrated moderate halflife, slow clearance and moderate bioavailablity in beagle dogs, identifying it as the first of a new class of orally bloavailable tissue-selective androgen receptor modulators.

so EXAMPLE 4 GTx-007 Analysts by HPLC A reversed phase high pressure liquid chromstograph (!PLC) assay was

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0 developed to quantitat GTx-.007 concntrations in dog plasma. Dog blood samples were obtained by vanipuncture and ccntifged at I 00Og for 15 minutes. Samples were stored frosen at -20"C until analysis. Individual samples wereon rapidly thawed and an aliquot ml) was spiked with intermal standard (20 pl of a 200 pg/ml aqueous solution of 00 s CM-U-87). An aliquot of 1 ml of acetonitrile was added to the samples to precipitate en plasma proteins. The samples were vortexed and than contrifuged at 1000g for tn minutes. The supernatant was decanted into glass extracdon tubes and 7.5 ml of ethyl 0 N acetate was added. The extraction mixture was left at room temperature for 20 minutes, o and vortexed several times during this interval. The samples were then centrifuged at 0o 1000g for 10 minutes, and the organic phase was removed and placed in conicalbottomed glass tubes. The organic phase was evaporated under nitrogen. The samples we reconstituted in 200 pi of mobile phase (35:65 acetonitile-water) and transferred to an autosampler vial for HPLC injection (Waters 717 plus autossampler, Waters Corp., Milford, MA). The isocratic mobile phase of 35% acetlonitrile in water was is pumped at a flow rate of 1 mlmin (Model 510, Waters Corp.). The stationary phase was a CI8 reversed phase column (Novapak CIS, 3.9 x 150 mm). Analytes ware monitored withUV detection at 270 nm (Model 486 absorbance detector, Waters Corp.). Retention times for GTx-007 and CM-II-87 were 11.1 and 16.9 minutes, respectively.

Chromatography data was collected and analyzed using Mllenmium software. Plasma concentrations of OTxlO-7 in each sample were determined by comparison to calibration curves. Calibration curves were construoted by adding known amounts of GTx-007 to dog plasma. Final OTx-007 concentrtions in dog plasma samples used in the calibration curves were 0.08, 0.2, 0.4, 2, 4, 10, and 20 pg/mItl. Calibration curves were linear over this concentration ange and exhibited correlation coefficients (r2) of 0.9935 or greater. Intra- and inter-day coefficients of variation for the standards ranged from 6.4% for 0.08 pgrm to 7.9% for 20 pg/nl.

Melting points were determined on a Thomas-Hoover capillary melting point apparatus and are uncorrected. aInftared spectra were recorded on a Perin Elmer Systermn 2000 FT-IR. Optical rotations were detemined on an Antopor UI Automtic Polarimter (Rudolph Research Model MI-589-10, Fairfield, New tersey). Proton and carbon-1l3 magnetic resonance spectra were obtained on a Indr AX 300 spectrometer

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C (300 and 75 MHz for 'H and respectively). Chemical shift values were reported as Sparts per million relative to totametbylsilane (TMS). Spectral data were consistent with assigned futtures. Mass spectra were determined on a Brker-HP Esquire LC System. Blemental analyses were perfonmed by Atlantic Microlab Inc. (Norcross, GA), s and found values wer within 0.4 of the thoretical values. Routine thin-layer Schromatography (TLC) was performed on silica gel on aluminum plates (silica gel 60 F tn 254, 20 x 20 cm, Aldrich Chemical Company Inc., Milwaukee, WI). Flash O chromatography was performed on silica gel (Merck, grade 60, 230-400 mesh, 0 Tetrahydrofura (THF) was dried by distillation over sodium metal. Acetonitrile (MeCN) and methylene chloride (CHaCl2) were dried by distillation from P 2 0s.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

Claims (5)

1. A selective androgen receptor modulator compound having in-vivo androgenic and anabolic activity of a nonsteroidal ligand for the androgen receptor, the selective androgen receptor modulator compound represented by the structure of formula II below, 00 O O YT wherein X is 0; Z is a hydrogen bond acceptor, NOz, CN, COR, CONHR; Y is a lipid soluble group; R is an alkyl or aryl group or OH; and Q is acetamido, trifluoracetamido, alkylamines, ether, alkyl, N-sulfonyl, 0-sulfonyl, alkylsulfonyl or carbonyl.

2. The selective androgen receptor modulator compound of claim 1, wherein X is 0, Z is N02, Y is a lipid soluble group, and Q is acetamido.

3. Use of the compound of claim 1 or 2 for the preparation of a medicament for use in hormone therapy in a subject.

4. Use of the compound of claim 1 or 2 for the preparation of a medicament for use in treating a hormone-dependent condition in a subject

5. Use of the compound of claim 1 or 2 for the preparation of a medicament for use in treating prostate cancer in a subject. 53 COMS ID No: ARCS-178323 Received by IP Australia: Time 14:03 Date 2008-02-07 O O 0, Dated this 12th day of April 2006 UNIVERSITY OF TENNESSEE RESEARCH FOUNDATION By their Patent Attorneys I 5 GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia In O 0 c,