WO2007048097A2 - Anbrosteeone derivatives and method of use thereof - Google Patents

Abstract

The disclosure provides androsterone derivatives. The derivatives of the disclosure are useful in the treatment of androgen- and estrogen-associated diseases and disorders,, including breast cancer.

Description

ANDROSTERONE DERIVATIVES AND METHOD OF USE

THEREOF CROSS REFERENCE TO KELATED APPLICATIONS

[0001] This application claims priority to Provisional Application Serial No. 60/729,463, filed October 20, 2005, the disclosure of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] The present invention was fimded in part by Grant No. DAMD17-99- 1-9018 awarded by DOD. The government may have certain rights in the invention.

FIELD OF THE INVENTION

[0003] The disclosure relates to novel steroids and more particularly to androsterone derivatives useful as anti-cancer, anti-obesity, anti-diabetic and hypolipidemic agents.

BACKGROUND

[0004] Dehydroepiandorsterone (DHEA) and dehydroepiandrosterone-sulfate are major adrenal secretory products in many mammalian species. Although DHEA- sulfate is the main precursor of placental estrogen and is converted into active androgens in peripheral tissue, there is no strong biological role for either DHEA or DHEA-sulfate in the normal subject. Several studies suggest that these steroids are associated with cell proliferative disorders as well as other androgen-associated diseases and disorders.

[0005] Examples of androgen-associated diseases and disorders include, but are not limited to, prostate cancer, benign prostatic hyperplasia, acne, seborrhea, hirsutism, androgenic alopecia, precocious puberty, adrenal hyperplasia, and polycystic ovarian syndrome. In addition, estrogen-associated diseases and disorders can be included such as, for example, breast cancer, endometriosis, leiomyoma, and precocious puberty.

SUMMARY

[0006] The disclosure provides androsterone derivatives, methods of synthesis and methods of use in the treatment of various androgen- and estrogen-associated diseases and disorders.

[0007] In one aspect, the androsterone derivatives inhibit breast cancer cell growth via counteracting the effect of female hormones and/or binding on receptors for such hormones (e.g., as antagonists) inducing inhibition of cellular proliferation and inducing killing of cells having cell proliferative disorders associated with androgens and estrogens.

[0008] In another aspect, the disclosure provides androsterone ester compounds, pharmaceutical compositions of these androsterone derivatives, methods of using androsterone derivatives (e.g., for the treatment of cancer). [0009] In yet another aspect, the disclosure provides androsterone- camptothecin combination compounds, pharmaceutical compositions of these androsterone derivatives, methods of using androsterone derivatives (e.g., for the treatment of cancer).

[0010] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and. advantages will be apparent from the description and drawings, and from me claims.

BRIEF DESCRIPTION OF THE FIGURE

[0011] Figure 1 shows the effect of an androsterone derivative of the disclosure on cancer cells.

DETAILED DESCRIPTION

[0012] As used herein and in the appended claims, the singular forms "a," "and, " and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antigen" includes a plurality of such, antigens and reference to "the immune cell" includes reference to one or more immune cells known to those skilled in the art, and so forth,

[0013] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. [0014] The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

[0015] Androstenone is a steroid hormone excreted in urine that reinforces masculine characteristics having the general formula as set forth in Formula 1:

[0016] The disclosure provides androsterone derivatives having the general formulas II and III:

[0017] In one aspect the R groups are aromatic (eg., aromatic nitrogen- containing heterocycles). In some aspect, the R groups have extended aromatic systems with electron withdrawing groups (e g., electrophilic). Aromatic nitrogen- containing heterocycles, typically contain a 5- or 6-membered monocyclic subsequent, or a tricyclic fused or linked 5- or 6-membered ring, such as imidazolyl, indolyl, pyridinyl, pyrimidinyl, pyrrolyl, quinolinyl, tetrazolyl, 1,2,4-triazolyl , and the like.

[0018] Aromatic nitrogen-containing heterocycles include, by way of example, 2-amino-pyridine, benzimidazole, 2,5-diaminopyridine, 2,4- dimethylimidazole.2,3-dimethylpyridine, 2,4-dimethylpyridine, 3,5- dimethylpyridine, imidazole, methoxypyridine, γ-picoline .2,4,6-trimethylpyridine, and combinations thereof.

[0019] In another aspect, the R group is a non-aromatic nitrogen-containing heterocyIe. Non-aromatic nitrogen-containing heterocycles, typically contain 4- to 6- membered rings such as acetimido, morpholinyl, lactams and imides (e.g., γ- butyrolactam, ε-caprolactam, N-phenyl-β-proprolactam), phteaelimido, piperidyl. piperidino , piperazinyl, pyrrolidinyl, succinimido , and the like. Non-aromatic nitrogen-containing heterocycles include, by way of example, 12-dimethylpiperidine, 2,5 dimethylpiperazine, 1,2-dimethylpyrrolidine, 1-ethylpiperidine, n- methylpyrrolidine, morpholine, piperazine, piperidine, pyrrolidine, 2,2,6,6- tetramethylpiper- idine, 2,2,4-trimethylpiperidine , and combinations thereof. In some more particular aspect, the R group is an atropine or a scopolamine. [0020] In another aspect, the methods of the disclosure utilize heterocyclic compounds in the synthesis of the androsterone derivatives of the disclosure. [0021] In yet another aspect, the methods of the disclosure utilize camptothecin compounds in the synthesis of the androsterone derivatives of the disclosure.

[0022] In one aspect of the disclosure, the androsterone derivative comprises a compound having the general formula II:

wherein R is selected from the group consisting of:

wherein * indicates the (+) chiral center in the original molecule.

[0023] In another aspect of the disclosure, the androsterone derivative comprises a compound having the general formula III:

wherein R is selected from the group consisting of:

[0024] One aspect of the disclosure is a pharmaceutical composition useful for treating an androgen-associated disease in a warm-blooded animal, which composition comprises compound of the disclosure as defined herein in combination with a pharmaceutically acceptable excipient. The composition is prepared in accordance with known formulation techniques to provide a composition suitable for oral, topical, transdermal,, rectal, by inhalation, parenteral (intravenous, intramuscular, or intraperitoneal) administration, and the like. Detailed guidance for preparing compositions of the disclosure are found by reference to the 18.sup.th or 19.sup.th Edition of Remington's Pharmaceutical. Sciences, Published by the Mack Publishing Co., Easton, Pa. 18040.

[0025] Unit doses or multiple dose forms are contemplated, each offering advantages in certain clinical settings. The unit dose would contain a predetermined quantity of active compound calculated to produce the desired effect(s) in the setting of treating disease. The multiple dose form may be particularly useful when multiples of single doses, or fractional doses, are required to achieve the desired ends. Either of these dosing forms may have specifications that are dictated by or directly dependent upon the unique characteristic of the particular compound, the particular therapeutic effect to be achieved, and any limitations inherent in the art of preparing the particular compound for treatment of cancer. [0026] The compound may be administered orally in a suitable formulation as an ingestible tablet; a buccal tablet, capsule, caplet elixir, suspension, syrup, trouche, wafer, lozenge, and the like. Generally, the most straightforward formulation, is a tablet or capsule (individually or collectively designated as an "oral dosage unit"). Suitable formulations are prepared in accordance with a standard formulating techniques available that match the characteristics of the compound to the excipients available for formulating an appropriate composition. [0027] The form may deliver a compound rapidly or may be a sustained- release preparation. The compound may be enclosed in a hard or soft capsule, may be compressed into tablets, or may be incorporated with beverages., food or otherwise into the diet.

[0028] The suitable formulation of an oral dosage unit may also contain: a binder, such as gum tragacanth, acacia, com starch, gelatin; sweetening agents such as lactose or sucrose; disintegrating agents such as com starch, alginic acid and the like; a lubricant such as magnesium stearate; or flavoring such a peppermint, oil of wintergreen or the like. Various other material may be present as coating or to otherwise modify the physical form of the oral dosage unit. The oral dosage unit may be coated with shellac, a sugar or both. Syrup or elixir may contain the compound, sucrose as a sweetening agent, methyl and propylparabens as a preservative, a dye and flavoring. Any material utilized should be pharmaceutically-acceptable and substantially non-toxic. Details of the types of excipients useful may be found in the nineteenth edition of "Remington: The Science and Practice of Pharmacy," Mack Printing Company, Easton. Pa, See particularly chapters 91-93 for a ful ler discussion. [0029] A compound, may be administered parenterally, e.g., intravenously, intramuscularly, intravenously, subcutaneously, or interperitonically. The carrier or excipient or excipient mixture can be a solvent or a dispersive medium containing, for example, various polar or non-polar solvents, suitable mixtures thereof, or oils. As used herein "carrier" or "excipient" means a pharmaceutically acceptable carrier or excipient and includes any and all solvents, dispersive agents or media, coating(s). antimicrobial agents, iso/hypo/hypertonic agents, absorption-modifying agents, and the like. The use of such substances and the agents for pharmaceutically active substances is well known in the an. Except insofar as any conventional media or agent is incompatible with the active ingredient, use in therapeutic compositions is contemplated. Moreover, other or supplementary active ingredients can also be incorporated into the final composition.

[0030] Solutions of the compound may be prepared in suitable diluents such as water, ethanol, glycerol, liquid polyethylene glycol(s), various oils, and/or mixtures thereof, and others known to those skilled in the art.

[0031] The pharmaceutical forms suitable for injectable use include sterile solutions, dispersions, emulsions, and sterile powders. The final form must, be stable under conditions of manufacture and storage. Furthermore, the final pharmaceutical form must be protected against contamination and must, therefore, be able to inhibit the growth of microorganisms such as bacteria or fungi. A single intravenous or intraperitoneal dose can be administered. Alternatively, a slow long term infusion or multiple short term daily infusions may he utilized, typically lasting from 1 to 8 days. Alternate day or dosing once every several days may also be utilized. [0032] Sterile, injectable solutions are prepared by incorporating a compound in the required amount into one or more appropriate solvents to which other ingredients, listed above or known to those skilled in the art, may be added as required. Sterile injectable solutions are prepared by incorporating the compound in the required amount in the appropriate solvent with various other ingredients as required. Sterilizing procedures, such as filtration, then follow. Typically, dispersions are made by incorporating the compound into a sterile vehicle which also contains the dispersion medium and the required other ingredients as indicated above. In the case of a sterile powder, the preferred methods include vacuum drying or freeze drying to which any required ingredients are added.

[0033] In all cases the final form, as noted, must be sterile and must also be able to pass readily through an injection device such as a hollow needle. The proper viscosity may be achieved and maintained by the proper choice of solvents or excipients. Moreover, the use of molecular or particulate coatings such as lecithin, the proper selection of particle size in dispersions, or the use of materials with surfactant properties may be utilized.

[0034] Prevention or inhibition of growth of microorganisms may be achieved through the addition of one or more antimicrobial agents such as chlorobutanol, ascorbic acid, parabens. thermerosal, or the like. It may also be preferable to include agents that alter the tonicity such as sugars or salts. [0035] In some cases, e.g., where a compound of the disclosure is quite water insoluble, it may be useful to provide liposomal delivery. The system restrains the compound of the disclosure by incorporating, encapsulating, surrounding, or entrapping the compound of the disclosure in, on, or by lipid vesicles or liposomes, or by micelles.

[0036] In addition, the disclosure provides methods of using the androsterone derivatives in the treatment of androgen-associate diseases and disorders including estrogen-associated diseases and disorders.

[0037] An androgen-associated disease or disorder includes, for example, prostate cancer, benign prostatic hyperplasia,, acne, seborrhea, hirsutism, androgenic alopecia, precocious puberty, adrenal hyperplasia, and polycystic ovarian syndrome. In addition, estrogen-associated diseases and disorders can be included such as, for example, breast cancer, endometriosis, leiomyoma, and precocious puberty. [0038] Precocious puberty is usually associated with an excess of androgen secretion, usually of adrenal origin. Current treatments include a blockade of adrenal secretion by glucocorticoids. Another treatment is the use of LHRH agonists to cause medical castration.

[0039] Polycystic ovarian syndrome is associated with an excess of androgen secretion by the ovaries. LHRH agonists are used among other, as treatment to cause medical castration.

[0040] Androgenic and estrogenic activity may be suppressed by administering androgen receptor antagonists ("antiandrogens") or estrogen receptor antagonists ("antiestrogens"), respectively. See e.g. WO 94/26767 and WO 96/26201. Androgenic and estrogenic activity may also he reduced by inhibiting receptor activation using receptor antagonists, suppressing androgen or estrogen biosynthesis using inhibitors of engines that catalyze one or more steps of such biosynthesis or by- suppressing ovarian or testicular secretions by known methods. [0041] Both androgen-related and estrogen-related diseases and disorder may be treated with an androsterone derivative of the disclosure. Androgen-sensitive diseases are those whose onset or progress is aided by androgen activation of androgen receptors, and should respond favorably to treatment with an androsterone derivative of the disclosure because of the reduction of androgen biosynthesis that is achieved thereby. Estrogen-sensitive diseases (diseases whose onset or progress is aided by activation of the estrogen receptor) should also benefit because many androgens whose biosynthesis is suppressed by the compound(s) of the disclosure are precursors to estrogens, and the compound(s) may therefore reduce estrogen biosynthesis as well. Androgen-sensitive diseases include but are not limited to prostatic cancer, benign prostatic hyperplasia, acne, seborrhea, hirsutism, androgenic alopecia and polycystic ovarian syndrome. Estrogen-sensitive diseases include but are not limited to breast cancer, endometrial cancer, endometriosis, and endometrial leiomyoma.

[0042] In some aspects of the disclosure it may be desirable to inhibit estrogen activity while maintaining androgen activity, for example, breast cancer (and some other estrogen-sensitive diseases, e.g. ovarian cancer, uterine cancer, and endometrial cancer) respond favorably to androgens. Therefore, a compound which inhibits estrogen activity, and which is also androgenic, can be especially useful for the treatment of breast cancer and other diseases which respond negatively to estrogen and positively to androgen.

[0043] In some aspects of the disclosure it may be desirable to promote cancer cell death using androgen-camptothecin combination compounds wherein the camptothecin component exhibit additive anticancer activity. It has been shown that camptothecin inhibits topoisomerase, an enzyme that is required for its swiveling and relaxation of DNA during molecular events such as replication and transcription. [0044] The androsteroma derivatives in accordance with the disclosure can be utilized as part of a combination therapy with other strategies, which modulate androgen- or estrogen-associated diseases and disorders through other mechanisms, thus providing synergistic combinations. For example, a combination therapy can include an androsterone derivative of the disclosure in combination with an agent selected from the group consisting of LHRH agonists (see, e.g., U.S. Pat. No. 4,659,695 and 4,666,883); FLUTAMIDE (N-[4-nitro-3-(trifluoromethyl)phenyl)3-2- methyl propanamide), NILUTAMIDE, or CASODEX; antiestrogenic (e.g., EM-800 reported in PCT/CA96/00097; TAMOXIFEN ((Z)-2-[4-(1,2-diphenyl-1-butenyl)}- N,N-dimethylethanamine) and ICT 182780 (available from Zeneca. UK), TOREMIFENE (available from Orion-Farmos Pharmaceutical. Finland), DROLOXIFENE (Pfizer Inc., USA), RALOXIFENE (Eli Lilly and Co., USA), LY 335563 and LY 353381 (Eli Lilly and Co., USA), LODOXIFENE (SmithKline Beecham, USA), LEVORMELOXIFENE (Novo Nordisk- A/S, Denmark): TRILOSTANE (2α-cyano-4α,5α-epoxy-17β-hydroxyandrostan-3-one); inhibitors of testosterone 5-alpha-reductase (e.g., PROSCAR); an aromatase inhibitor (e.g., ARlMIDEX); and androgenic compounds (e.g., medroxyprogesterone acetate, and megestrol acetate).

[0045] In general, for both androgen-associated diseases and estrogen- associated diseases, simultaneous treatment with inhibitors of sex steroid biosynthesis (inhibitors of enzymes which catalyze one or more steps of estrogen or androgen biosynthesis), and with estrogen receptor antagonists and/or androgen receptor antagonists, are believed to have additive rather than redundant effect because they are acting in a beneficial manner by a different mechanism. [0046] Different sex steroid-dependent diseases respond differently to both androgen receptor activation and estrogen receptor activation. For example, breast cancer responds unfavorably to estrogen receptor activation, but favorably to androgen receptor activation. On the other hand, benign prostatic hyperplasia responds unfavorably to activation of either the estrogen or androgen receptor, [0047] When an androsterone derivative of the disclosure is used, either alone or as part of one of the combination therapies described herein, the attending clinician will typically target the subject's serum concentration between 0.5 ng/rnl and 100 ng/ml, more typically between 1 ng/ml and 20 ng/ml, and more commonly between 1 ng/ml and 10 ng/ml. Serum concentration may be measured by various techniques known in the art (e.g.. LC/MS). When administered orally, the dosage which is usually effective to provide the desired serum levels is between 1.0.mg and 1.000 mg of active ingredient per day per 50 kg of body weight, typically between 10 mg and 500 mg and more commonly between 10 mg and 100 mg. However, dosage will vary with the bioavailability of the chosen inhibitor and with individual subject's response. The attending clinician will typically monitor aa individual subject's response and metabolism and adjust the subject's dosage accordingly. When administered by injection, a lower dosage is typically used, e.g. 10 mg to 100 mg per day per 50 kg of body weight.

[0048] All of the active ingredients (including the androsterone derivatives of the disclosure) used in any of the therapies discussed herein may be formulated in pharmaceutical compositions which may include one or more additional active ingredients as discussed above. Alternatively, they may each be administered individually separately or simultaneously. In some embodiments of the disclosure, one or more active ingredients are formulated in a single pharmaceutical composition. [0049] The working examples below are provided to illustrate, not limit the disclosure. Various parameters of the scientific methods employed in these examples are described in detail below and provide guidance for practicing the disclosure in general.

EXAMPLES

[0050] The following examples are given to provide representative compounds included as part of this disclosure. The examples also provide descriptions of in vitro and in vivo assays to aid in determining the utility of the compounds. Throughout the examples chemical formulas will be used to name compounds as appropriate,

[0051] 1. (3β, 5a)-3-Hydroxyandrotan-17-one9,10-dihydro-9-10-dioxo -2 anthracenecarboxylate (990624). The reaction mixture of epiandrosferone (1.30 mg, 0.45 mmol), anthraquinone- 2-carbonyl chloride (135 mg, 0.5 mmol), triethylamine (100 mg, 1.0 mmol) and dichloromethan( 6.0 ml) were stirred at room temperature for 20h. Then 20 ml of dichloromethane was added. Organic layer was washed with water (20 ml), saturated NaHCO3 solution (15 ml) and brine (20 ml), and then dried over MgSO₄. After the solvent was removed in vacuo, the resulting solid was recrystallized from ethyl alcohol and ethyl acetate to give 46 mg (3β, 5α)-3- Hydroxyandrostan-17-one 9.10-dihydro-9,10-dioxo-2-anthracenecarboxylate. [ 0052 ] The chemical structure analysis was performed by ¹HNMR (CDCl₃, 600 MHz); δ 8.93 (s, 1H, Ar-H), 8.39-8.43 (m, 4H, Ar-H) 7.84 (s, 2H, Ar-H), 5.05 (s, 1H, H-3) 2.45 (t, 1H, H-16), 2.20-0.70 (m, 2.1H, ), 0.94 (s, 3H, CH₃- 19),0..88 (s, 3H, CH₃).

[0053] 2. (3β,5α)-3-Hydroxyandrostan-17-one 5-nitro-2-furoate (991027). The reaction mixture of 5-nitro-2-furoic acid (157 mg, 1.0 mmol ), epiandrosterone (163 mg , 0.60 mmol), EDCI (200 mg, 1.05 mmol ). DMAP (20 mg 0.2 mmol) and THF (4 ml) was stirred at room temperature for 6.5 h. Evaporation of THF gave a residue which was dissolved in dichloromethane (20 ml). The organic layer of dichloromethane was washed with H₂O, 5% Na₂CO₃, H₂O and brine, and then dried over Mg SO₄. The solvent was removed under vacuum. The resulting solid was recrystallized from C₂H₅OH to give 180 mg (73.2 %} (3β,5α)-3-hydroxyandrostan- 17-one 5-nitro -2-furoate 134 mg. [0054] The chemical structure analysis was performed by ¹I-Os⁷MR (CDCI_?. 600 MHz): 5735 (s, 1H₁ Ar-H), 7.28(s, IR Ar-H), 4.99 (s, 1H, H-3), 2.45 (t; 1H, H- 16), 2.2⁽M)J0 (m, 21H, ), 0.5X⁾ (s, 3H, CHH9)_.. 0.87 (β, 3H. CH₅)- £00553 X (3βρα)-3-Hy<li-oxyanc!rostan-J7-one (+)-2-(2^,6-tetπmiiro-9- flonrenylHlene- aminøøxy)|)roprionate (991022), The reaction mixture of (+)-2- (2,4s5.<j-tetranitro-9-floureny{idene-ammooxy)--propπorJ.ic add (224 rog, 0.5 mmol), epiandrøslarone (122 mg, 0.45 rπmoi), DCC (.123 mg, 0.6 nana!), DMAP (10 mg, 0.1 mmol) and THF (6 m\) was stirøtl at room temperature for 6 h. Then 4 drops of water were added to above solution. After stirred for K) nm, the mixture was filtered and the solid was washed with TBF. Evaporation of THF gave a residue which was dissolved in dichioromeihane (20 ml). The organic ϊavβi" of dichlorømeihane was washed with HχO, 5%

HoO and brine, wni then dried over MgSU4. The solvent was removed under vacuum. The resulting solid was recrysiallixed from ethyl acetate and petroleum ether to give 149 mg (47.3 %) (3p_?5α)-3- hydro\yandrostsn~17--on(-*-)-2-(2_t4_:,5.64etrai)itiO-9~Boureny!idenfi-aminoo^y)~ proprionatδ.

[ 00S 6 J The chemical siatctoe analysis was performed by ¹HNMR (CDCis, 600 MHz): S 9.59 (s, JH, Ar-H), 8.97 (d, 2H, Ar-H), 8.90 (s, 1H, Ar-H), 5.22 % IR CHCO), 4.88 (ra, IH_> CHO), 2.45 (t. 1H₇ H-16), 2.20-0.70 (m, 2iH, )_r 1.83 (d, 3H, CH3), 0.88 (s, 3H, CHrI9)_; 0.86 (s, 3H, CH₃). [0057] 4. l~<(3β,5α)-3-Ηyiϊϊmyandrosta»-17-o«eH-'<2- (hydros>^vi»ethyϊ)aiitiiϊra<|ui«one)-s«ccrøate (991120). The reaction, mixture of {3β,5α)-3-Mydraxyandroslarvl7~one monosuccinate (25 mg. 0.064 mmol), 2- (hydroxymethyl) aiϊthraquinone (16 rag, 0.067 mmol), EDCI (20 mg, 0,11 rninoi), DMA? (2 nig.0,02 mmol) and dichlofomethane (4 ml) was stirred at room temperature for 6 h. Then 20 ml of dichlαrαmethane was added, Organic layer was washed with HaO, 5% NaαCOs- H2O and brine, and then dried over MgSO*. The solvent was removed under vacuum to give 37 mg l-((3β,5α)-3-Hydroxyandrostan- 17-o.ne)~4-(2-(hydroλymethy]) ao.thraquinone)suc€inale as yellow solid. [005S] The chemical structure analysis was performed by ¹HNMR (CDCI₃, 600 MH*): δ O0(ni 4R Ar-H), 7.80 (ra, 3H_? Ar-H), 5.30 (s, 2a ArCH₂OX 4.65⁾ (rrt 1H, CHO), 2.74 (t 20, CH₂), 2.64 (t 2Ϊ1 CB₂), 2.4.1 (ra, IiI H₁₆), 2.20-0.67 (ra 2JH, ), 1.83 (d, 3H, CH3), 0.S2 (s, 3H, CH₃), 0.80 (s, 3H, CH₃). ,1-( )3-Hydroxyandrostan e) 5- nitroquinoline)succinate 991123 ).The re -

nitroquinoline ( ( 0mg, 011 mmol)- DMAP . mmol and dichloromethane was stirred at room t

e, e.g

Figure I). The derivatives were less toxic to normal cells and provided to be effective at .killing cancer ceils (particularly breast cancer cells).

Table h In vttrø antitumor activity of androsterotte analogues øgaiost HCTl 16 cells.

[00643 7. The compounds of this example are prepared utilizing a camptothecitt-based compound.

10065 ] 7A. Caaiptothecin-20-O-ester of 4-carboxyphenoxyacetic acid (intermediate contponnd). The mixture of campfcsthecin (100 mg, 0.287 mmoi), 4- carboxypheaoxj'acetic acid (S.12 mg, 0.57 tmnol), EDCI (82 mg, 0,43 ramolX 0MAP (10 mg. 0J mmoϊ), N.N-di.mβ&γIformamide (4 n\l) and dichlomraethaftβ (4 ml) were stirred in lhe room temperature for 48 h, then dicMoromethane (50 ml) was added to the solution. Organic layer was washed will* water (20 ml), saturated NaHCOs aqueous solution (20 nil) end bάne (20 mi), and then dried over MgSQφ After the solvent was removed trøder reduced pressure, the resulting solid was separated by column chromatography (eSuent: CHCU; CaHsOH 5:1) to afford 80 mg caroptøthecin- 20-O- 4-fluoropheno^χyacetate, yield; 67.0 %, rap "(dec). [0066] The chemical structure analysis was performed by ¹HNMR (CDCi₃. 600MHz); δ 8.40 (s, IH, Ar-H), 8.30 (d, IH, Ax-B% 8.07 {m, 2H, Ar-H), 7.95 (cl IH, Ar-B), 7,86 (t, 1H, Ar-H), 7.67 (t, 1H, Ar-H), 7.25 (s, IH, Ar-HX 6.96 (rø, 2H, Ar-H), 5.68 (4 1H, Hl 7), 5.43 ((1,1H, W 7), 5,29 <s, 2H, HS) , 4.91 {<}, 2H, OCH₃COK 2.25 (dm, 2H, CH₂), 0.97 (t 3H, CH₃). C 0 Q 67 ] 7B. Camptotbecin^0S-O- (4-carbøxy phenoxyaeetatε) iiakett with epfcmdrosterøne (0.103021). The mixture of canφt.ofoecm-2()$-0~(4~ carbo^φhenoxyacetete) (10 mg, 0.019 mmol), epiandrosterαne (11 nig, 0.038 πanol), EDCΪ (25 rag, 0.13 mmol), DMAP (2 mg, 0.02 mmol), and dichlorome&ane (3 ml) were stirred in the room temperature for 24 h, then diehiαramethane (20 ml) was added to the solution.

[ 00683 Organic layer was washed with water (20 mi), saturated NaHCOs aqueous solution (J0 ml) aid brine (20 mi), and then dried over MgSO*. After the solvent was removed under reduced pressure, the resulting solid, was separated by column chromatography (elusnt; ethyl acetate-elhanoi 9:1) to afford 2,0 mg target. compound.

£00691 The chemical stasctmre analysis was performed by ¹SINMR (CDCl:,, 600MH?)'. δ 8.41 (s_* 1H, Ar-H), 8.27 (<L 1H, Ar-H), 7.98 (m, 2H, Ar-H), 7.86 (i; .!H, Ar-H), 7,69 (t, 1H, Ar-H), 7.29 (s_? 1H, Af-H), 7.24 (s, 1H, Ar-H), 7.21 (s, 1H. Ar-B), 6.95 <d, JH, Ar-BX 5.68 <d, Ul, CPT-Hl 7), 5.42 (dJli CPT-HJ 7), 5.29 (>, 2H, CPT- H5) , 4.89 (q, 2H, OCH₂CO), 4.23 (m, 1H, βpiandrosterone-HS), 2.60 -L00 (m, 30H, CPT-HlS and epiandrosterone-H).

[0070] 1C, Camptothedn-20S-O~(4-c;arbβxyphe«βxj'accfate) fϊαked with a»drostero»e (010216), The mixture of caraptothecin~20S~Q-(4~ car^'bo^'phenoxyacetste) (10 m%.0.019 mmoi), androsterone {^'i 1 røg_r 0.038 πimol), EDCΪ (25 mg, 0. B mmol X DMAP (2 mg, 0.02 mmoi), and diehlørømethane (3 ml) were stirred in the room temperature for 24 b, then dieWαromethaae (20 ml) w'as added to the solution. Organic layer was washed with water (20 miχ saturated NaHCOs aqueous solution (10 ml) and brine (20 ml), and then dried over MgSO^ After the solvent was removed under reduced pressure, the resulting solid was separated by column chromatography (elυent: etbyl acetate-eihano! 9:1 ) to afford 4.3 mg solid.

C00713 The chemical structure analysis was performed by ¹HNMR (CDCI3. 600MHx): d 8.41 (s, 1H, Ar-H), 8.28 (i 1H, Ar-H)₃7.99 (m, 3H, Ar-H), 7.87 (t, 1H, Ar-H), 1.69 (t. 1H, Ar-H), 7.35 (s. 1H, Ar-H), 7.21 (s, 1H, Ar-H), 6.96 (d, 2H₁, Ar-H), 5,71 (4 1H, CPT-H17X 5.43 (d,lH, CPT-H17), 5.29 (_S> 2H, CPT-H5) , 4.90 (q_? 2H_? OCH₂CO), 4.1.3 (m, IU, atidrosterone-i-B), 3.00-2.000», 21H, CPT-Hl 8 and androstøooe-H), 1.28 (s_t 6H_S CH₃), 0.97 <t, 3H. CPT-Hlθ). 10072] Many camptothecin-based compounds are generally available in the art and would be known to one of ordinary skill in the srl. Some examples of such camptothecin-based compounds include: (20S)~9-πitro CFl': (20S)-7-chIoro-n~ propyldiroethyisilyl CPT; {20S)-10-hydroxy-7-ch}oro-n-pfopyldimethylsilyt CPT; (20S)-H>acetoxy-7<^hioιo~n"propyldimeιhylsily! CPT; (2()S)-7"tert>- butyldimeihylsilvl CPT; (20S)-lO-hydroxy-7-teri-but>-ldiitieUiylsi1yl CPT; (20S)-IO- acetoxy-7-teii-butyldimethylsilyl CPT; (20S)-9-hydroxy CPT: (20S)-9-amino CPT; (20S)-10-amino CPT; (20S)-9-ammo-l ⁽Hydroxy CPT; (20S)-9-meΛylainino CPT; (20S)-9-chloro CPT; (20S)-9-fluoro CPT; (20S)-9-piperidino CPT; (20S)-O- moφhoiinomeihyl CPT; (20SH>,10-dϊchloro CPT; (20S)-U⁾'biOmo CPY; (20S>10- chloio CPT; (20S)-ii>-methyI CPT: (20S)-10-flιrøro CPT: (20S)-l<>mtro CPT; (20S)- 10,H-methylcnediox\' CPT: (2OS)-I0-foimyi CPT; (20S)-10-nonyicarbomloxy CPT; (20$)-!()-«ndec\'lcarbonyloxy CPT; (20S)-I O-heptadewlcarbonyloxy CPT; (20S)-I O-iionadecykarbonyloxy CPT; (20S)-9-nitro-]0.ll-methylenedioxy CPT; (20S)-9-<4-metlwlpipera/inylmetiiy I)-I «-hy droxT (CPT); (20S)-9-|4-(l -piperidijw)- 1 - piperidiτrønMhyl|-K⁾-hydroxy CPT; (20S)-9-methyl-?0,π-nκ;%lenedio,\y CPT; (20S)-^-chloro-IOJ t-meibvlenedioxy CPT; (20S)-^-CVaHO-IOJ I -melhyienedioxy CPT; (20S)-y-acetox\ -l0J ϊ-methyleπedioxy CPT; (20S)-9-acet>'laiiiino-10J 1- raetl\ylenediox>' CPT; (20S)-9-aminomethyl-i04wdroxy CPT; (20S)-9-edioxymethyl- 10-hydroxy CPT; (20S)-9-nieihyiafninomethyi-?0-hydTOχv CPT; (20S)-9-n- propylammomelhyl-10~b>'drøxy CFT: (20S)-9-cUjnelln'lamJnonielhyl-10-hydiO_Axy CPT; (20$)-9-cyclohexylaiτιinomethyi-10-bydfθxy CPT: (20S)-9-(2- hydroΛyethyl)arainometbyI-10-hydroxy CPT; (2()S)-9-(uimethylammorAio)niet})y)-i()- hydro.vy CPT. metbancsulfonate; (20S)-9-morpbolinomelhyI-lO-hydroxy CPT; (20S)- 9-cyanomethyl-lO-hv dro.vy CPT; (20S)-CPT-7-a1dehyde; (20S)-10-niethoxy CPI -7- aidehyde; (20S)-7^«acetoxyraethyl CPT; (20S)-7-acetoxymethyl-iO-me{iiy! CFT; (20S)-7-c> ano-10-melhoxy CPT: (20S)-7-cyaϊio CPT; (20S)-7-forniyIethenyI CPT; (20S)-7-etboxycarl>onyle{henyl CPT: (20$>7-cyaooeihenyl CPT; (20$)-7-(2> dicyanoethe^-1) CPT; (20S>7-(2-cyano-2-ethoxycarbony})ethenyl CFF; (20S)-7- ethoxycarbonylelhyl CPT; (20S)-7-etliyl CTT; (20S)-7-n-propyl CPT; (20S)-7- acetoxvmelhyl CPT; (20S)-?-n-prθiyϊcarbonyioxymethy! CPT; (20S)-7- eOioxycaτbonyl CPT; (20S)-7-eih.y1-I O-hydroxy CPT; (20S)-7-eihyi-10-acelylo>Λ' CPT; (20S)-7-inethyl-iO-aminocarbonyloxy CPT; (2OS)-7-n-propyl-I0- piperidinocazbonyloxy CPT; (20S)-7-etfayl-10-(2-dimetliyianύao)e{iiyl CPT: and

1? (2(>SH-eUt>-|'10-carbamoyloxy derivatives of CPT such as (20S)-7-elhyl-10-H(l- piperidino)-piperidino carbonyloxy CPT; (20S)-7-ethyl-IO^^'l~pϊperazine)carbony1oxy CPT: (20S)-7-ethyl-l 0-(4-i-propylaminocarbonylroethylpiperazine)carbonyloxy CPT; (20S)-7-etIiyH0-|4(I-pyrrolicIiny0piperaϊ!ine]carbonvloxy CPT; (20S)-7-ethyl-10- I(4-(dJmethylanύnιo)-l-piperidmo|carbonyloxy CPT: (20S>7~etlwl-iO-|4>(di-n-> propy)amino)-l-piperidinol]c«rbon.yloxy CPT: (20S)-7~ethyl-lu-l(4-(di~n~ bυtylaminoH -piperidinoicar bonyloxy CPT; <20S)-7-etli\ 1- 10-{ 4-( 1 -py rrolidino)- 1 - piperidiπo)jcarbonyloxy CPT; (2αSh7-etliyl-10-|^"4-(l-pjperidino)-l- piperidinolc{«tonylox\ CPT: (2()S)-7-eihyl-lO-|N-meihyl-N--J- (dimethylanΩno)eUiyIajrøιo|carbonyloxy CPl^'; (20S)-7~(_itert-bιrtyklime%lsilyl)Cl^rf; (20S>7-{{eit-butoxyimtnoinethyl) CPT (Gimatecan): (2ϋS)-7-butyl-10_fl 1- methvlenedioxy CPT; {20S)-7-bromorøetliyl-K)-hydroxy CPT; (20SV7-bulγl-H)- amino CPT; (20S)-7>(ier{-butyldimethylsi{yl)-IO-hydrox>- CPT; (2OS)-7-j{2- trimeihylsilyl)cthyj)| CPT (Rarenfican); (2⁽>S)-7-|(4-f1uorophcnoxy>aceti'loxymethyl] CPT; (2⁽⁾S)-7-|(4-methoxyphenoxy)acetyloxymethyl| CPT; (20S)-7-K4-cyaiio-3- fluorophenoxy)acetylox>-meU\yI| CPT; (20S)-7-|(M,5- trimeiiio.xypihenyl)ac€^{y1oxN'me%i| CPT; <20S)-l⁽M(4-cyano-3- fluorophenoxy)acetyloxyj CPT: (20S)-10-|(3.4,5-trimethoxyphenyi)aceiyloxy] CPl'; (20S)-7-(4-me(hylpipera/inoiτielhy!e£ϊe)-10J 1-ethylenedioxy CPT (Exatecan); (20S)- 7-j_.2-(N-isopropylamno)eU^l} CPT (Belotecan); (20S)-|5(RS)-(2-hydrox\'elhc»n')J CPT (20; and combinations thereof.

[ 0073 ] The compounds of this example 8τe prepared by reacting any androsterone aaialog or any epiandrosierone an∑ilog.

[ 00743 One of skill in the art will recognize that other similar aiidrosterone analog or epiandrosterone analog may be obtained from commercial sources or prepared by art-recognized procedures to be used in these steps to prepare compounds of this disclosure. By reacting a compound shown in the list of camptolhecin-based analogs with an androsterone analog in accordance with the guidelines for reaction condition, compounds of the disclosure will be obtained. These compounds will exhibit the desired characteristics to a greater or lesser extent [ 0075] To test the effect of androsterone derivati ves of this example. HCTl 16 cells (colorectal carcinoma cells) were plated in 60 mm Petri dishes containing 2.7 ml of medium (modified McCoy's 5a medium containing 10% fetal bovine serum and

100 units/ml penicillin and 100 Og/ml streptomycin). The cells were incubated in a

18 CO?, incubator at 3TC for 5 hours for attachment to the bottom of Petri dishes. Drugs were made up fresh in medium at ten times the fma! concentration, and then 0.3 mi of tliis stock solution was added to the 2.7 mi of medium containing 5% bovine calf serum (BCS) in the dish. The cells were then incubated with drugs for 72 hours at 37'⁵C. At the end of incubation the drug-containing media were decanted, the dishes were rinsed with 4 ml of Hank's Balance Sail: Solution (HBSS), 5 ml of fresh roediu.ni containing 15% BCS was added, and the dishes were returned to the incubator for colony formation. The cell colonies stained with methylene blue (0.3% in ethanol) were counted using colony counter after incubation for 8 days for HCTl 16 cells. Cell survival was calculated and the values of .IC 50 (the drug concentration producing 50% inhibition of colony formation) were determined for each tested compound. Hie values of ΪC50 were 2.5 nM for 010216 and 3.S iM for 0100321. The results are presented in Table 2,

TABLE 2t CeIi survival of HCTU6 ceils treated with 01.0216 and

010302!

[0G76] A number of embodiments have beers described. Nevertheless, ii will be understood that various modifications may be made without departing from the spirit and scope of the description. Accordingly, other embodiments are within the scope of the following claims.

\ 9

Claims

What is claimed h

.1. A compound comprising ihe general formula U:

Il wherein R is selected .frora ihe group consisting of an aromatic, an aromatic nitrogen- coriiarrang heterocycie, and a non-aromatic nitrogen-containing . helerocycle.

2. The compound of claim 1. wherein the R group has an aromatic system wiϊh electron withdrawing groups.

3. The compound of claim L wherein ihe aromatic nitrogen-containing heteroeycle is a 5- or 6~membered monocyclic substHueflt or a bic>^!cllc fused or Unked 5- or 6-merabered ring,

4. Hie compound of claim 3, wherein fhs aromatic nitrogen-containing heiefocycie is an imidazolyj. an indolyi. a -pyridmyf, apyrimidiayl. a pyrrolyf, a. qumoHnyl, a tetrazoly), and a i^^-lriaxolyl.

5. The compound of claim 3. wherein ilιe aromatic mtrogen-coataining hεierocv'tie is selected from fee group consiling 2-aπ?iπo~p>^fHdine, benzimidazole, 23-diarainopyridine, 2,4-dim8thylimidaxo1e_? 2,3-dirae%lpyridin8, 2,4- dimethyl pyridines 3,5-dimethylpyridine, imidazole, methoxypyridine. γ-picoline, and 2,4,6-trimethylpyridine.

6. The compound of claim i , wherein the non-aromatic nitrogen-containing heterocyle comprises a 4- to 6-membered rings selected from the group consisting of an acetimido, a morpholinyl, a lactam, an imide,a phthalimido , apiperidyl, a piperidino, a piperazinyl, a pyrrolidinyl, and a succinimido.

7. The compound of claim 6, wherein the imide is selected from the group consisting of γ-butyrolactam a-caprolactam, N-phenyl-propiolact am.

8. The compound of claim 1 , wherein t he non-aromatic nitrogen-containing heterocycle is selected from the group consisting of a 1,2-dimethyl piperidine, a 2,5- dimethylpiperazine , a 1,2-dimethylpyrrolidine -a 1-ethylpiperidine, an- methylpyrrolidine, a morpholine, a piperazine, a piperidine, a pyrrolidine, a 2,2,6-6- tetramethylpiperidine, and a 2.2,4-trimethylpiperidin e.

9. The compound of claim 1 , wherein the R group is an atropine or a scopolamine.

10. The compound of claim 1 , wherein the R group is a camptothecin analog.

11. The compound of claim 1, wherein the R group is selected from the group consisting of:

14. The compound of claim 11. herein the aromatic nitrogen-coøiainirig heterocyde Is a 5- or 6~.membered monocyclic substiluenf or a tricyclic fused or linked 5- or 6-membered ring.

1.5. The compound of claim 13, wherein the aromatic mtrogeft-cαntaimng heterαcycie is an mύdazoiyL an mdolyl, apyrkiinyL apyriraidmyl, apyrrolyl, a qumolmyi. a lelrøzolyl, and a 1,2,4-tnazolyJ.

.

16. The compound of claim O, wherein the aromatic nitrogen-containing hetsrocycle is selected from the group conshing 2-amino~pyriditte_> berizirπidazrole, 2,5-diaminopyridine, 2,4-dimethylimidazole, 2;3-dime&ylpyridine, 2.4- dimetfeylpyridiae, 3.5-dimethylpyridine, ijnidaxoϊe, .methoxy pyridine, γ-picoli«e_» mid 2_t4.6-trimeihy1pyridine.

17. The compound of claim 11₈ wherein the non-aromatic nitrogen-containing heterøcyle comprises a 4- to 6-membered rmgs selected from the group consisting of an acetimido, a raorpholinyl, a lactam, an tmi<1e, a phthaJirøkio. a piperidy). a pipsrkϋπo, a piperazinyl, a pyrroIidinyL and a succirύmido.

18. The compound of claim i(_\ wherein the imide is selected from the group consisting of y-bαiyroiactam,. s-ciϊproiactam- M-phenyl-β-propioiactarn.

19. The compound of claim 11 , wherein the non-aromatic nitrogen -containing heterocyclic is selected from the group consisting of a l_t2-dimethylpiperidine_t a 2,5- dimefliylpiperaxmβ, a LS-dimethylpyrroiidine, a 1-ethylpiperidine, a »- methylpyrrolidine, a niorpholine, a piperazine, a pipβridine, a pyrrolidine, a 2,2,6,6- letnarøetaylpipefidine, and a 2_f2_;4-tjfime£hy.lpiperidine.

20. I'he compound of claim 12. wherein the R group is an atropine or a scopolamine,

2 i . The compound of claim 12, wherein (he R group is a camptøthεem analog.

22. The compound of claim 1.2, wherein R is selected .from the group consisting of:

23. A pharmaceutical composition; comprising the compound of any one of claims 1-22 m a pharmaceutical Jy acceptable carrier.

24. A method of treating an androgen -associated disease <>.?^■ disorder or an estrogen-associated disease or disorder comprising administering to a subject in need of such treatment a therapeutically effective amount of s compound of aay one of claims 1-22.

25. The method of claim 24, further comprising administering to the subject a therapeutically effective amount of an agent selected from the group consisting of art antiestrogen or an aαtiaαdrogen.

26. A method of treating or reducing the risk of developing breast cancer comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound of any one of claims 1-22 or a pharmaceutical composition thereof.

27. The method of claim 25, further comprising administering to the subject a therapeutically effective amount of an antiestrogen agent

28. The method of claim 2CL further comprising administering a therapeutically effective amount of an LHRH agonist or antagonist

29. The method of claim 26, further comprising administering a therapeutically effective amount of a second androgenic compound.