WO2009005839A2 - Compounds and methods of use - Google Patents

Abstract

Compounds comprising a structure represented by formula (I): X - L - Z wherein X is a steroid hormone receptor binding moiety; Z is (i) a moiety that binds to an extranuclear element or (ii) a blocking moiety that disrupts the interaction between a steroid hormone receptor and a steroid hormone receptor coactivator; and L is a linking group covalently bound to X and Z, wherein the linking group is physiologically-noncleavable and has a structural length and rigidity sufficient to allow the compound to bind to the steroid hormone receptor and (i) the extranuclear element or (ii) the blocking moiety.

Description

COMPOUNDS AND METHODS OF USE

This application claims the benefit of U.S. Provisional Application No. 60/958,351 , filed July 3, 2007, which is incorporated by reference in its entirety.

Field

The present disclosure relates to compounds that may bind to hormone receptors, pharmaceutical compositions that include these compounds, and methods for inhibiting cancer, such as hormone-resistant cancer, and steroid hormone-associated diseases.

SUMMARY

Disclosed herein are compounds comprising a structure represented by formula I:

X - L - Z

wherein X is a steroid hormone receptor binding moiety;

Z is (i) a moiety that binds to an extranuclear element or (ii) a blocking moiety that disrupts the interaction between a steroid hormone receptor and a steroid hormone receptor coactivator; and

L is a linking group covalently bound to X and Z, wherein the linking group is physiologically-noncleavable and has a structural length and rigidity sufficient to allow the compound to bind to a steroid hormone receptor and (i) the extranuclear element or (ii) the blocking moiety.

Also disclosed herein are compounds comprising a structure represented by formula II:

X - alkynylene linker - Z wherein X is a steroid hormone receptor binding moiety; and

Z is (i) a moiety that binds to an extranuclear element or (ii) a blocking moiety that disrupts the interaction between a steroid hormone receptor and a steroid hormone receptor coactivator.

In certain embodiments Z of formula I or formula II is a colchicinoid.

In certain embodiments Z is a tubulin binding moiety.

According to a further embodiment, disclosed herein are compounds comprising a structure represented by formula III:

W - alkynylene linker - Y

wherein W is selected from cyanonilutamide, nilutamide, flutamide, hydroxy fiutamide, bicalutamide, megestrol acetate, bazedoxifene, clomifene, fulvestrant, raloxifene, tamoxifen, toremifene, mifepristone, andarine, ostarine, prostarin, andromustine, BMS-564929, spironolactone, eperenone, ketoconazole, or an analog thereof; and

Y is a colchicinoid or a taxane.

Also described herein is a pharmaceutical composition comprising a therapeutically effective amount of the compound of formula I, II or HI and a pharmaceutically acceptable carrier.

The compound of formula I, II or III may be administered to a subject for inhibiting in the subject a disease associated with and dependent upon hormone receptor activity or lack of activity.

The compound of formula I, II or III also may be administered to a subject for inhibiting in the subject a neoplasm, particularly neoplasms that have developed a resistance to chemotherapeutic (such as taxane) treatment. A further disclosure involves a method for anchoring a steroid hormone receptor in cell cytoplasm that includes contacting the cell with a compound of formula I or II, thereby binding the compound to an element in the cell cytoplasm. In certain embodiments the compound is bound to tubulin in the cell cytoplasm.

An additional disclosed method for binding a steroid hormone receptor binding agent to tubulin includes contacting a compound of formula I or II with tubulin, thereby binding the compound to the tubulin.

The foregoing will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structures of combretastatin A-4, colchicine, thiocolchicine, nilutamide, cyanonilutamide and a novel compound disclosed herein ("CCN").

FIG. 2 depicts a synthesis scheme for a novel compound disclosed herein. FIG. 3 represents a possible mode of CCN binding to the androgen receptor after molecular dynamics equilibration in which binding occurs along channel I, which is the path between helices 3, 6, 7, and 1 1.

FIG. 4 represents another possible mode of CCN binding to the androgen receptor after molecular dynamics equilibration in which binding occurs in the path along channel II, which resides between helices 1 1 and 12. X-ray structure is shown in gray colors. FIG. 5 is a graph showing the results of an assay to determine inhibition of tubulin assembly by CCN. Assembly of 10 μM tubulin was induced by 0.8 M glutamate +0.4 mM GTP. The solution was preincubated for 15 min. at O⁰C prior to addition of GTP, with assembly followed for 20 min. at 30⁰C. Assembly was measured turbidimetrically by change in apparent absorbance at 350 nm. Concentrations of CCN are indicated in μM. FIG. 6 are Western blots of nuclear and cytoplasmic LNCaP extracts shown after 1 1 hours of exposure to vehicle (Veh), 0.1 μM nilutamide (Nilut), 0.1 μM colchicine (Colch) or 0.1 μM CCN (CCN). Ponceau S stained membrane is shown for protein loading control. Colchicine and CCN increase cytoplasmic AR protein levels without a detectable change in nuclear AR. FIG. 7 is a graph depicting cell toxicity data. LAPC4AI cells (androgen independent prostate cancer cells) were plated at 50,000 cells per well in a 96 well plate, exposed to vehicle, 0.1 uM, 0.05 uM or 0.01 uM colchicine or CCN or 0.05 colchicine plus 0.05 uM nilutamide for 48 hours and a cell titer blue survival assay was done. Details of this assay can be found on the company protocol pdf (http://www.promega.com/tbs/tb317/tb317.pdf). The experiment was done in triplicate with error bars representing one standard deviation. The experiment was repeated and a representative experiment is shown. The data shows that 0.05 uM CCN is clearly more toxic to androgen independent prostate cancer cells that 0.05 uM colchicine and more toxic than 0.05 uM colchicine plus 0.05 uM nilutamide.

FIG. 8a is graph showing that CCN inhibits dihydrotestosterone (DHT) - induced AR luciferase activity, even when CCN is present at much lower concentrations than DHT. FIG. 8b is a graph showing that CCN does not effectively inhibit dexamethasone (dex) - induced glucocorticoid receptor (GR) activation. This suggests that CCN is specifically targeted to AR, as opposed to other steroid hormone receptors.

FIG. 9a is a graph showing the inhibition of CCN on AR transcriptional activity using an MMTV-luciferase construct. MMTV is a promoter that is responsive to AR as well as GR. FIG. 9b is a graph showing that CCN inhibits AR on a second androgen-responsive promoter (ARE4). This data shows that CCN inhibition of AR is generalizable to more than one androgen-responsive promoter. The AR inhibition activity at these concentrations is much greater than any AR antagonist that is FDA approved and currently used clinically for prostate cancer.

FIG. 10a is a graph depicting tubulin polymerization induced by various compounds as measured by a turbidimetry assay. The curve for vehicle is the lowest curve, paclitaxel is highest curve (until it intersects with the curve for VT192-219), and the four middle curves are four synthetic taxanes linked to cyanonilutamide. Compound VTl 92-219 is second highest curve until it intersects with the curve for paclitaxel and then it becomes the highest curve. FIG 10b depicts the structure of compound, VT 192-219. Cyanonilutamide is conjugated to the taxane moiety through the 10-position with a rigid alkyne linker, similar to the colchicine-cyanonilutamide compound.

FIG. 1 1 depicts a gene expression analysis in LAPC4 prostate cancer cells of compound 219, paclitaxel, compounds 85, 106, 105, 297, 300, nilutamide and vehicle control done in triplicate. The region of the starkest contrast in gene expression among the samples is shown. Among the genes that are induced significantly by paclitaxel and compound 85 and to a much lesser extent with the other compounds is survivin. FIG. 12 is a chart showing the cell growth inhibitory effect of CCN against the NCI 60 cell line. This data shows that CCN has growth inhibitory effects across multiple cancer cell lines that occurs as laws as the nanomolar range.

DETAILED DESCRIPTION

"Administration of and "administering a" compound should be understood to mean providing a compound, a prodrug of a compound, or a pharmaceutical composition as described herein. The compound or composition can be administered by another person to the subject (e.g., intravenously) or it can be self-administered by the subject (e.g., tablets).

"Alkyl" refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, H-propyl, isopropyl, w-butyl, isobutyl, f-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. A "lower alkyl" group is a saturated branched or unbranched hydrocarbon having from 1 to 10 carbon atoms.

"Alkoxy" refers to a radical of the formula -OR, wherein R is an alkyl.

"Alkoxycarbonyl" refers to a radical of the formula -C(O)OR, wherein R is an alkyl.

"Alkynylene" refers to a divalent organic radical composed primarily of carbon and hydrogen atoms and containing at least one carbon-carbon triple bond. In certain embodiments, the alkynylene group includes 2 to 12 carbon atoms, more particularly 2 to 8 carbon atoms.

"Analog," "Derivative" or "Mimetic": An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, or a change in ionization. An analog may or may not be synthesized or derived from the parent compound. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28). A derivative is a biologically active molecule derived from the base structure. A mimetic is a molecule that mimics the activity of another molecule, such as a biologically active molecule. Biologically active molecules can include chemical structures that mimic the biological activities of a compound. An "animal" is a living multicellular vertebrate organism, a category that includes, for example, mammals and birds. A "mammal" includes both human and non-human mammals. "Subject" includes both human and animal subjects.

The term "aralkyl" refers to an aryl group having an alkyl group, as defined above, attached to the aryl group. An example of an aralkyl group is a benzyl group.

"Aryl" refers to an aromatic group having the ring structure characteristic of phenyl, naphthyl, etc. (i.e., either the 6-carbon ring of benzene or the condensed 6-carbon rings of the other aromatic derivatives). The term "aromatic" also includes "heteroaryl group," which is defined as an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorous. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylic acid, or alkoxy, or the aryl group can be unsubstituted. The term "alkyl amino" refers to alkyl groups as defined above where at least one hydrogen atom is replaced with an amino group.

"Carboxyl" refers to a -COOH radical.

The term "cycloalkyl" refers to a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term "heterocycloalkyl group" is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorous.

"Estrogenic agent" refers to a compound, or a pharmaceutical composition containing such compound or agent, that is, or is inclusive of, any of the sex steroid hormones (or natural or synthetic analogs thereof) referred to generically under the name "estrogen." Such estrogenic agents generally include, but are not limited to, naturally occurring estrogens [estradiol (E₂), estrone (Ei), and estriol (E₃)], synthetic conjugated estrogens, polyestradiol phosphate, sulfated estrogens, and selective estrogen receptor modulators (SERMs). Illustrative specific estrogenic agents include 2-methoxyestradiol (also known as l,3,5(10)-estratriene-3,17βdiol 2 methyl ether); 17β-estradiol (also known as estra-1, 3,5(10)-triene-3, 17β-diol); and SERMs such as clomiphene; cycladiene; tamoxifen; nafoxidine; nitromifene citrate (N-55,945-27); 13-ethyl- 17.alpha.-ethynl-17.beta.-hydroxygona-4-9-l 1-trie- n-3-one (R2323); diphenol hydrochrysene; erythro-MEA; allenolic acid; cyclofenyl; chlorotrianisene; ethamoxytriphetol; triparanol; CI- 626; CI-680; MER-25; U-1 1 ,555A; U-1 1,10OA; ICI-46,669; ICI-46,474; CN-55,945; the triphenyl compounds described in U.S. Patent No. 2,914,563; and benzothiophenes such as those described in U.S. Patent No. 5,624,940. See, Gruber et al., "Production and actions of estrogens" N Engl J Med 346(5):340-52, 2002. "Oestrogen" is recognized as an alternative spelling for "estrogen," and as such the term "estrogenic agent" as used herein is inclusive of oestrogen.

"Extranuclear element" refers to any substance (e.g., an enzyme or protein), or structure (e.g., organelle) that is located within a cell but outside of a cell nucleus, and that performs a cellular function. The extranuclear element may be present in the cytoplasm or the cell membrane. The extranuclear element does not enter the cell nucleus, or is more abundant in the cytoplasm as compared to the nucleus. In certain embodiments, extranuclear elements reside only in the cytoplasm.

"Hormone-resistant cancer" refers to cancer that progresses despite administration of hormonal therapy (such as androgen deprivation therapy, androgen receptor antagonists and adrenal ablation for prostate cancer). For example, prostate cancer and breast cancer are both hormone-dependent cancers, which become resistant to hormonal therapy by reactivation of their respective steroid receptors - androgen receptor (AR) and estrogen receptor (ER). Castrate-resistant prostate cancer is synonymous with hormone-resistant prostate cancer, hormone-refractory prostate cancer, and androgen-independent prostate cancer.

The term "hydroxyalkyl" refers to an alkyl group that has at least one hydrogen atom substituted with a hydroxyl group. The term "alkoxyalkyl group" is defined as an alkyl group that has at least one hydrogen atom substituted with an alkoxy group described above.

"Inhibiting" or "treating" refers to inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as hormone-resistant cancer. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. As used herein, the term "ameliorating," with reference to a disease, pathological condition or symptom, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of relapses of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology. By the term "coadminister" is meant that each of at least two compounds be administered during a time frame wherein the respective periods of biological activity overlap. Thus, the term includes sequential as well as coextensive administration of two or more drug compounds.

"Pharmaceutically acceptable salts" of the presently disclosed compounds include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N- benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts may be prepared by standard procedures, for example by reacting the free acid with a suitable organic or inorganic base. Any chemical compound recited in this specification may alternatively be administered as a pharmaceutically acceptable salt thereof. "Pharmaceutically acceptable salts" are also inclusive of the free acid, base, and zwitterionic forms. Descriptions of suitable pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002). "Pharmaceutically acceptable complexes" of the presently disclosed compounds include those complexes or coordination compounds formed from metal ions. Such complexes can include a ligand or chelating agent for bonding with the compounds.

A "pharmaceutical agent" or "drug" refers to a chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject. Optionally substituted groups, such as "optionally substituted alkyl," refers to groups, such as an alkyl group, that when substituted, have from 1-5 substituents, typically 1 , 2 or 3 substituents, selected from alkoxy, optionally substituted alkoxy, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, aryl, carboxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, halogen, optionally substituted heteroaryl, optionally substituted heterocyclyl, hydroxy, sulfonyl, thiol and thioalkoxy. In particular, optionally substituted alkyl groups include, by way of example, haloalkyl groups, such as fluoroalkyl groups, including, without limitation, trifluoromethyl groups. A "steroid hormone receptor coactivator" refers to substances that physically associate with steroid receptors and are required for steroid receptor function. Examples of steroid hormone receptor coactivators include steroid receptor co-activator 1 (SRCl), transcriptional intermediary factor 2 (TIF2), Tat interactive protein (Tip60), and CREB binding protein (CBP). A "therapeutically effective amount" refers to a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this may be the amount of a compound disclosed herein useful in inhibiting or treating hormone-resistant cancer in a subject. Ideally, a therapeutically effective amount of an agent is an amount sufficient to inhibit or treat the disease without causing a substantial cytotoxic effect in the subject. The therapeutically effective amount of an agent will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition.

"Type I steroid hormone receptors" are intracellular receptors that perform signal transduction for steroid hormones, and that have an associated heat shock protein. "Type II steroid hormone receptors" are located in the cell nucleus and can bind to nonsteroid ligands such as thyroid hormones and/or vitamin A.

The above term descriptions are provided solely to aid the reader, and should not be construed to have a scope less than that understood by a person of ordinary skill in the art or as limiting the scope of the appended claims. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The word "comprises" indicates "includes." It is further to be understood that all molecular weight or molecular mass values given for compounds are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All chemical compounds disclosed herein include both the (+) and (-) stereoisomers (as well as either the (+) or (-) stereoisomer), and any tautomers thereof.

Prostate cancer is the leading cause of nonskin malignancy in men. Growth and survival of prostate cancer is dependent on androgens and androgen receptor (AR) signaling. Therefore, advanced disease is generally first treated with androgen deprivation therapy by medical or surgical castration. Metastatic disease almost always overcomes androgen deprivation and progresses as castrate-resistant prostate cancer (CRPC). A wealth of evidence suggests that CRPC is still reliant on AR for tumor cell survival and disease progression. In the castrate-resistant setting, AR is reactivated by a variety of mechanisms that include but are not limited to AR gene amplification and other mechanisms of increasing AR expression, and ligand-independent activation by growth factors and cytokines. Furthermore, a subset of androgen-responsive genes are reactivated in CRPC, and prostate-specific antigen declines will often occur with secondary hormonal therapies that also target AR. Together, this evidence suggests that AR is still a valid target in CRPC, and compounds that have novel AR-targeting mechanisms should provide new avenues for prostate cancer therapy.

The novel and inventive rationale for the design and synthesis of a compound that has independent extranuclear element-binding and steroid receptor-binding moieties as described herein is four-fold. First, many extranuclear element-binding, especially tubulin-binding, drugs are used for cancer chemotherapy. In fact, the only form of chemotherapy shown to prolong survival for metastatic prostate cancer patients is a tubulin binding drug. Addition of a steroid receptor-binding moiety to a therapeutic agent could selectively target steroid receptor- expressing cancer cells, with minimal impact on cells that do not express the steroid receptor. Second, the nuclear import of steroid hormone receptors is a microtubule-dependent process. The use of a colchicinoid, which only binds to the soluble tubulin heterodimer and disrupts tubulin polymerization, may thereby inhibit the nuclear import of the steroid hormone receptor. Third, independent steroid hormone receptor-binding and extranuclear element-binding moieties in a single compound would potentially result in concomitant steroid hormone receptor binding and extranuclear element binding, thereby anchoring the steroid hormone receptor to the extranuclear element. Thus, if the extranuclear element does not enter the nucleus the bound steroid hormone receptor would remain in the cytoplasm. However, a difficulty is preservation of binding to the hydrophobic steroid hormone receptor ligand-binding domain, in which the ligands are completely buried. Adding a linker or a bulky moiety to a steroid hormone receptor ligand could therefore easily lead to loss of steroid hormone receptor-binding activity. Fourth, if steroid hormone receptor ligand-binding activity can be conserved with a compound that has a linker that extends outside the ligand binding domain, this could disrupt binding to steroid receptor coactivators that are required for steroid hormone receptor function. The novel compounds disclosed herein are targeted for inhibiting the activity of intracellular steroid receptors, particularly Type I steroid receptors. In certain embodiments, the novel compounds disclosed herein can bind to a steroid hormone receptor via a first moiety and/or an extranuclear element via a second moiety. The hormone receptor-binding moiety is structurally independent of the extranuclear element-binding moiety. In other words, the compounds are "bifunctional" in the sense that they include the first binding moiety and the second binding moiety. Extranuclear elements reside only in the cytoplasm or the cell membrane. Although not bound by any theory, it is believed that the binding of the bifunctional compound to a steroid hormone receptor and the extranuclear element anchors the receptor to the cytoplasm, thus inhibiting nuclear localization (i.e. movement of the receptor from the cytosol to the cell nucleus upon activation) and function of the receptor.

The modality utilized with the novel compounds is not a straightforward strategy since it involves very unpredictable molecular interactions. For example, the hydrophobic ligand- binding domain is buried deep within the steroid receptor meaning there is tremendous potential for steric hindrance.

In one illustrative embodiment, the novel compound is a conjugate or construct of a colchicinoid or a taxane and an AR antagonist (e.g., cyanonilutamide) designed to inhibit AR function in hormone-resistant prostate cancer. A problem in multifunctional AR-binding compounds is steric hindrance of binding to the embedded hydrophobic AR ligand-binding pocket. Despite the bulky side chain projecting off of the AR-binding moiety, one synthesized example of the novel conjugates binds to AR with a K, of 449 nM. Structural modeling of this synthesized compound in the AR ligand-binding domain using a combination of rational docking, molecular dynamics and steered molecular dynamics simulations reveals a basis for how this compound, which has a rigid alkyne linker, is able to bind to AR. Surprisingly, it has been found that this compound also binds to tubulin and inhibits tubulin function to a greater degree than colchicine itself. The tubulin-inhibiting activity of this compound increases cytoplasmic AR levels in prostate cancer cells. Finally, it has been found that this compound has greater toxicity against androgen independent prostate cancer cells than the combination of colchicine and nilutamide. Together, these data point to several ways of inhibiting steroid receptor function in hormone-resistant cancer.

The hormone receptor binding moiety and can be any moiety that binds, or may bind, to the steroid hormone receptor of interest and deactivates or inhibits the receptor's response in a disease mechanism or biological cascade. Steroid hormone receptors that could be targeted include the Type I steroid hormone receptors such as the sex hormone receptors (androgen, estrogen, progesterone), glucocorticoid receptors, and mineralocorticoid receptors; and the Type II steroid hormone receptors such as vitamin A receptor, vitamin D receptor, retinoid receptor, and the thyroid hormone receptor. Illustrative agents that could be used as the precursor for the hormone receptor binding moieties include androgen receptor antagonists, estrogen receptor antagonists, progesterone receptor antagonists, selective androgen receptor modulators (SARMs), selective estrogen receptor modulators (SERMs), estrogenic agents, androgenic agents, glucocorticoids, mineralocorticoids, and any antagonists thereof. Illustrative androgen receptor antagonists include cyanonilutamide, nilutamide, flutamide, hydroxyflutamide, bicalutamide, megestrol acetate, spironolactone, and analogs thereof. Illustrative SERMS include bazedoxifene, clomifene, fulvestrant, raloxifene, tamoxifen, toremifene, and analogs thereof. Illustrative progesterone receptor antagonists include mifepristone and analogs thereof. Illustrative SARMS include andarine, ostarine, prostarin, andromustine, BMS-564929, and analogs thereof. Illustrative mineralocorticoid antagonists include spironolactone, eperenone, and analogs thereof. Illustrative glucocorticoid antagonists include RU486 (mifepristone), ketoconazole, and analogs thereof.

The extranuclear element-binding moiety can be any moiety that binds, or may bind, to an extranuclear element. Illustrative examples of specific extranuclear elements to which the moiety could bind include tubulin, actin, FK binding protein, cyclophilin, cellular lipid or cellular sugar.

The tubulin binding moiety can be any moiety that binds, or may bind, to tubulin. The moiety could bind to monomeric or multimeric forms of tubulin, including microtubules and microtubule-associated proteins. Suitable moieties can be identified, for example, from tubulin- binding assays such as those described in Hamel, Cell Biochem Biophys. 2003;38(l): 1-22. The binding moiety can be derived from a microtubule depolymerizing agent (e.g., colchicinoid) or a microtubule stabilizing agent (e.g., taxane). Illustrative agents that could be used as the precursor for the tubulin binding moiety include the colchicinoids (colchicine, thiocolchicine, and combretastatin A-4 which all bind to a common site on tubulin known as the colchicine site; colchicinoids are also referred to as colchinoids), the vinca alkaloids (vinblastine, vincristine, vinorelbine), the taxanes (paclitaxel, docetaxel), nocodazole, podophylotoxin, rhizoxin, epothilones A and B, dolastatin, phenstatin, highly oxygenated derivatives of cis- and trans- stilbene and dihydrostilbene, steganacin, curacin A, 2-methoxyestradiol, dihydroxy- pentamethoxyflavanone, sanguinarine, griseofulvin, cryptophycin, chelidonine, and analogs thereof. In certain particular embodiments, the tubulin binding moiety is a colchicinoid. In other embodiments, the tubulin binding moiety is a taxane. Illustrative agents that could be used as the precursor for the actin-binding moiety include dolastatin-1 1, bistramide-A, latrunculin A, latrunculin B, doliculide, cytochalasin, phalloidin, and analogs thereof. Illustrative agents that could be used as the precursor for the cyclophilin-binding moiety include cyclosporin and analogs thereof. Illustrative agents that could be used as the precursor for the FK protein-binding moiety include FK506 and sirolimus. A physiologically-noncleavable linker was selected to allow for the possibility of concomitant steroid receptor and extranuclear element binding. "Physiologically-noncleavable" means that the linker does not undergo cleavage (i.e., neither the steroid hormone receptor binding moiety nor the extranuclear element binding moiety are released) under the intracellular or extracellular conditions in the cell environment of interest. More particularly, the linker will not cleave in cellular chemical conditions (e.g., salt or electrolyte concentration, metabolite concentration, pH, osmolality, dielectric constant, temperature, pressure, etc.) or enzymatic conditions.

The linker may have sufficient length to extend the extranuclear element-binding moiety outside the steroid receptor ligand-binding domain. For example, the linker may be at least about 10 angstroms, more particularly about 10-10.5 angstroms.

The linker includes an alkynylene group or a structurally similar group that provides the desired rigidity. In certain embodiments, the linker may also include an ether and/or ester functional group. For example, the linker may have a structure of:

-CH₂-O-C=C-;

-C(O)O-C≡C-; or

R^l0-C(O)O-C≡C- , wherein R¹⁰ is -C(O)-(CH₂)_X- or -C(O)-(CH₂)₂-C(O)-(O-(CH₂)₂)_y-O-C(O)-(CH₂)₂)-, wherein x is 1 to 20, 1 to 10, or 2 to

5 and y is 1 to 100, 2 to 100, 2 to 50, or 2 to 20. According to other illustrative embodiments, the linker may have a structure of:

-(C(R')₂)_n-C≡C-(C(R²)₂)_m-

wherein R¹ and R² are each independently H, alkyl, aryl, hydroxyl, alkoxy, carboxyl, alkoxycarbonyl, or halogen; and n and m are each independently an integer from 0 to 5. More particularly, the structure may be:

-(CH₂)_n-C≡C-(CH₂)_m- ;

-(C(R³)H)_n-C≡C-(CH₂)_m-

wherein R³ is alkyl, aryl, hydroxyl or halogen;

-(CH₂)_n-C≡C-(C(R³)H)_m-

wherein R³ is alkyl, aryl, hydroxyl or halogen;

-(CH₂)_a-(C(R³)H)_b-C≡C-(CH₂)_c-(C(R³)H)_d-

wherein R³ is alkyl, aryl, hydroxyl or halogen, a + b = 0 to 5, and c + d = 0 to 5.

Illustrative alkynylenes include ethynylene, 1-propynylene, 2-propynylene, 1- butynylene, 2-butynylene, 3-butynylene, 1 -pentynylene, 2-pentynylene, 3-pentynylene, 4- pentynylene, 1-hexynylene, 2-hexynylene, 3-hexynylene, 4-hexynylene, 2,4-hexadiynylene, 5- hexynylene, 1-heptynylene, 2-heptynylene, 3-heptynylene, 4-heptynylene, 5-heptynylene, 6- heptynylene, 1-octynylene, 2-octynylene, 3-octynylene, 4-octynylene, 5-octynylene, 6- octynylene, and 7-octynylene.

In certain embodiments, the linker does not include a salicylamide N-Mannich base structure or the linker moiety is not attached to either the steroid hormone receptor binding moiety or the extranuclear element binding moiety via a salicylamide N-Mannich base structure. The linker group structure may be bonded directly to the steroid hormone receptor binding moiety and directly to the extranuclear element binding moiety without the need for any intervening or intermediate spacer atoms or further functionalities. In other words, only a covalent bond is present between the linker group structure and the steroid hormone receptor binding agent structure, and only a covalent bond is present between the linker group structure and the extranuclear element binding agent structure.

According to another embodiment, the steroid hormone receptor binding moiety can be linked (via the linking group) to a blocking moiety that disrupts the interaction between a steroid hormone receptor and a steroid hormone receptor coactivator. In other words, the blocking moiety is a structure that acts as a wedge between the steroid hormone receptor and the steroid hormone receptor coactivator thus inhibiting their interaction. The blocking moiety can be an alkyl, aryl, alkoxy, alkoxycarbonyl, cycloalkyl, heterocycloalkyl, heteroaryl, hydroxyalkyl, alkoxyalkyl or an aralkyl. The blocking moiety is covalently bonded to the linking group. The extranuclear element to which the compound with an extranuclear element- binding moiety binds also may disrupt the interaction between a steroid hormone receptor and a steroid hormone receptor coactivator.

Illustrative compounds are shown below:

wherein n is 0 to 20, more particularly 3 to 20; X is a halogen (e.g., Cl or F), OCH₃ or N₃; and R is an aryl (e.g., a 6-member ring such as phenyl or benzyl) or heterocyclic group such as a 5-member ring (e.g., furyl, thienyl, or pyrryl), or OR' wherein R' is CpCi₂ straight-chain or branched alkyl. The three structures shown immediately above all incorporate a polyethylene glycol moiety and an alkynylene in the linker structure.

Examples of specific compounds are:

1 IC₅₀=O.113uM VT192-183

2 IC₅₀=0.323uM VT192-204

3 IC₅₀=5.403uM VT192-210

9 IC₅₀=0.004uM VT206-105

11 IC₅₀=9.532uM VT206-92

These compounds may be synthesized by techniques known in the art. Examples of the synthesis of these compounds are described below in detail. The agents listed above may be "precursors" of the hormone receptor binding moiety or the extranuclear element binding moiety in the sense that the chemical structure of the agent may be modified or activated so that it can be covalently bound to the linking group. The linker may be attached to the hormone receptor binding moiety in such a manner that the linker is directed in the correct orientation allowing it to follow a channel out of the steroid receptor, and activity intrinsic to the steroid hormone binding moiety is not lost. The linker may be attached to the extranuclear element- binding moiety in such a manner that the binding activity is not lost.

The compound disclosed herein can be used to inhibit, treat or prevent any type of disease associated with and dependent upon hormone receptor activity or lack of activity. Such diseases include hormone-resistant (i.e., hormone-independent) cancers, neoplasms or diseases. Hormone-resistant cancers include hormone-resistant prostate cancer, hormone-resistant breast cancer, and hormone-resistant endometrial cancer. The compounds can also be used as the primary treatment for prostate cancer, breast cancer and endometrial cancer. In other embodiments, the compounds can be used to inhibit, treat or prevent precursors to invasive cancers, such as ductal carcinoma in situ (a breast cancer precursor), lobular carcinoma in situ (a breast cancer precursor), or prostate intraepithelial neoplasia (a precursor to prostate cancer). The compounds also can be used to target androgen receptors in skin/hair follicles for treating androgenetic alopecia and acne, androgen receptors in the prostate for prostatic hyperplasia and androgen receptors elsewhere in the body that are responsible for symptoms and disease processes related to hypogonadism, such as osteoporosis, cardiovascular disease, muscle wasting, anemia and sexual dysfunction. These compounds also can be used to target estrogen receptors to treat endometriosis, polycystic ovarian syndrome and symptoms and disease processes related to female hypogonadism or menopause, such as osteoporosis, cardiovascular disease, sexual dysfunction and hot flashes. In addition, these compounds may be used to modify sex steroid receptors for the use of contraception and for the treatment of infertility. Furthermore, these compounds may be used for cancers that have a biology related to glucocorticoid function, such as leukemias, lymphomas and multiple myeloma. They may also be used for a deficiency or excess or thyroid function by modifying thyroid receptor function, a deficiency or excess of vitamin D by modifying vitamin D receptor and a deficiency or excess of vitamin A by modifying vitamin A receptor. They may also be used to modify vitamin D receptor or vitamin A receptor for the prevention of cancer. The compound may also be used for the treatment of hypertension by the modification of glucocorticoid receptor and mineralocorticoid receptor. In particular, for treating hormone-resistant prostate cancer, the hormone receptor binding moiety, for example, can be selected from steroidal agonists such as testosterone and dihydrotestosterone; steroidal antagonists such as cyproterone acetate, nonsteroidal antagonists such as nilutamide, flutamide and bicalutamide; and SARMS such as andarine, ostarine, prostarin and andromustine. For treating hormone-resistant breast cancer, the hormone receptor binding moiety can be selected from steroidal estrogens such as estradiol; SERMS such as tamoxifen; and estrogen receptor antagonists such as fulvestrant.

The compounds disclosed herein may be included in pharmaceutical compositions (including therapeutic and prophylactic formulations), typically combined together with one or more pharmaceutically acceptable vehicles or carriers and, optionally, other therapeutic ingredients (for example, antibiotics or anti-inflammatories). The compositions disclosed herein may be advantageously combined and/or used in combination with other antiproliferative therapeutic agents, different from the subject compounds. In many instances, co-administration in conjunction with the subject compositions will enhance the efficacy of such agents. Exemplary antiproliferative agents include cyclophosphamide, methotrexate, adriamycin, cisplatin, daunomycin, vincristine, vinblastine, vinarelbine, paclitaxel, docetaxel, tamoxifen, flutamide, hydroxyurea, and mixtures thereof.

Such pharmaceutical compositions can be administered to subjects by a variety of mucosal administration modes, including by oral, rectal, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to other surfaces. Optionally, the compositions can be administered by non-mucosal routes, including by intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, intrathecal, intracerebroventricular, or parenteral routes. In other alternative embodiments, the compound can be administered ex vivo by direct exposure to cells, tissues or organs originating from a subject. To formulate the pharmaceutical compositions, the compound can be combined with various pharmaceutically acceptable additives, as well as a base or vehicle for dispersion of the compound. Desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like. In addition, local anesthetics (for example, benzyl alcohol), isotonizing agents (for example, sodium chloride, mannitol, sorbitol), adsorption inhibitors (for example, Tween 80), solubility enhancing agents

(for example, cyclodextrins and derivatives thereof), stabilizers (for example, serum albumin), and reducing agents (for example, glutathione) can be included. Adjuvants, such as aluminum hydroxide (for example, Amphogel, Wyeth Laboratories, Madison, NJ), Freund's adjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL-12 (Genetics Institute, Cambridge, MA), among many other suitable adjuvants well known in the art, can be included in the compositions. When the composition is a liquid, the tonicity of the formulation, as measured with reference to the tonicity of 0.9% (w/v) physiological saline solution taken as unity, is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced at the site of administration. Generally, the tonicity of the solution is adjusted to a value of about 0.3 to about 3.0, such as about 0.5 to about 2.0, or about 0.8 to about 1.7.

The compound can be dispersed in a base or vehicle, which can include a hydrophilic compound having a capacity to disperse the compound, and any desired additives. The base can be selected from a wide range of suitable compounds, including but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (for example, maleic anhydride) with other monomers (for example, methyl (meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers, such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives, such as hydroxymethylcellulose, hydroxypropylcellulose and the like, and natural polymers, such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often, a biodegradable polymer is selected as a base or vehicle, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters and the like can be employed as vehicles. Hydrophilic polymers and other vehicles can be used alone or in combination, and enhanced structural integrity can be imparted to the vehicle by partial crystallization, ionic bonding, cross-linking and the like. The vehicle can be provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres and films for direct application to a mucosal surface.

The compound can be combined with the base or vehicle according to a variety of methods, and release of the compound can be by diffusion, disintegration of the vehicle, or associated formation of water channels. In some circumstances, the compound is dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) prepared from a suitable polymer, for example, isobutyl 2-cyanoacrylate (see, for example, Michael et al., J. Pharmacy Pharmacol. 43: 1 -5, 1991 ), and dispersed in a biocompatible dispersing medium, which yields sustained delivery and biological activity over a protracted time. The compositions of the disclosure can alternatively contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. For solid compositions, conventional nontoxic pharmaceutically acceptable vehicles can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

Pharmaceutical compositions for administering the compound can also be formulated as a solution, microemulsion, or other ordered structure suitable for high concentration of active ingredients. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity for solutions can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of the compound can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. In certain embodiments, the compound can be administered in a time release formulation, for example in a composition which includes a slow release polymer. These compositions can be prepared with vehicles that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system or bioadhesive gel. Prolonged delivery in various compositions of the disclosure can be brought about by including in the composition agents that delay absorption, for example, aluminum monostearate hydrogels and gelatin. When controlled release formulations are desired, controlled release binders suitable for use in accordance with the disclosure include any biocompatible controlled release material which is inert to the active agent and which is capable of incorporating the compound and/or other biologically active agent. Numerous such materials are known in the art. Useful controlled- release binders are materials that are metabolized slowly under physiological conditions following their delivery (for example, at a mucosal surface, or in the presence of bodily fluids). Appropriate binders include, but are not limited to, biocompatible polymers and copolymers well known in the art for use in sustained release formulations. Such biocompatible compounds are non-toxic and inert to surrounding tissues, and do not trigger significant adverse side effects, such as nasal irritation, immune response, inflammation, or the like. They are metabolized into metabolic products that are also biocompatible and easily eliminated from the body. Exemplary polymeric materials for use in the present disclosure include, but are not limited to, polymeric matrices derived from copolymeric and homopolymeric polyesters having hydrolyzable ester linkages. A number of these are known in the art to be biodegradable and to lead to degradation products having no or low toxicity. Exemplary polymers include polyglycolic acids and polylactic acids, poly(DL-lactic acid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), and poly(L-lactic acid-co-glycolic acid). Other useful biodegradable or bioerodable polymers include, but are not limited to, such polymers as poly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid), poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta- hydroxy butyric acid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethyl methacrylate), polyamides, poly(amino acids) (for example, L-leucine, glutamic acid, L-aspartic acid and the like), poly(ester urea), poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides, polysaccharides, and copolymers thereof. Many methods for preparing such formulations are well known to those skilled in the art (see, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978). Other useful formulations include controlled-release microcapsules (U.S. Patent Nos. 4,652,441 and 4,917,893), lactic acid-glycolic acid copolymers useful in making microcapsules and other formulations (U.S. Patent Nos. 4,677, 191 and 4,728,721) and sustained-release compositions for water-soluble peptides (U.S. Patent No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterile and stable under conditions of manufacture, storage and use. Sterile solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the compound and/or other biologically active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders, methods of preparation include vacuum drying and freeze-drying which yields a powder of the compound plus any additional desired ingredient from a previously sterile-filtered solution thereof. The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

In accordance with the various treatment methods of the disclosure, the compound can be delivered to a subject in a manner consistent with conventional methodologies associated with management of the disorder for which treatment or prevention is sought. In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of the compound and/or other biologically active agent is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and/or ameliorate a selected disease or condition or one or more symptom(s) thereof. Typical subjects intended for treatment with the compositions and methods of the present disclosure include humans, as well as non-human primates and other animals. To identify subjects for prophylaxis or treatment according to the methods of the disclosure, accepted screening methods are employed to determine risk factors associated with a targeted or suspected disease of condition (for example, hormone-resistant cancer) as discussed herein, or to determine the status of an existing disease or condition in a subject. These screening methods include, for example, diagnostic methods, such as various ELISA and other immunoassay methods, which are available and well known in the art to detect and/or characterize disease-associated markers. Risk of breast cancer can be determined, for example, by family history, genetic testing or a combination of risk factors using clinical risk models, such as the Gail model (^"Lancet Oncol. 2002 Oct;3(10):61 1-9). Risk of prostate cancer can be determined, for example, by PSA measures, including absolute PSA levels, PSA velocity, other PSA measures, levels of various hormones, cytokines and growth factors, family history, genetic testing and ethnicity (J Clin Oncol. 2005 Jan 10;23(2):368-77). These and other routine methods allow the clinician to select patients in need of therapy using the methods and pharmaceutical compositions of the disclosure. The administration of the compound of the disclosure can be for either prophylactic or therapeutic purpose. When provided prophylactically, the compound is provided in advance of any symptom. The prophylactic administration of the compound serves to prevent or ameliorate any subsequent disease process. When provided therapeutically, the compound is provided at (or shortly after) the onset of a symptom of disease or infection. For prophylactic and therapeutic purposes, the compound can be administered to the subject in a single bolus delivery, via continuous delivery (for example, continuous transdermal, mucosal or intravenous delivery) over an extended time period, or in a repeated administration protocol (for example, by an hourly, daily or weekly, repeated administration protocol). The therapeutically effective dosage of the compound can be provided as repeated doses within a prolonged prophylaxis or treatment regimen that will yield clinically significant results to alleviate one or more symptoms or detectable conditions associated with a targeted disease or condition as set forth herein. Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject. Suitable models in this regard include, for example, murine, rat, porcine, feline, non- human primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models (for example, immunologic and histopathologic assays). Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the compound (for example, amounts that are effective to elicit a desired immune response or alleviate one or more symptoms of a targeted disease). In alternative embodiments, an effective amount or effective dose of the compound may simply inhibit or enhance one or more selected biological activities correlated with a disease or condition, as set forth herein, for either therapeutic or diagnostic purposes.

The actual dosage of the compound will vary according to factors such as the disease indication and particular status of the subject (for example, the subject's age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the compound for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental side effects of the compound and/or other biologically active agent is outweighed in clinical terms by therapeutically beneficial effects. A non-limiting range for a therapeutically effective amount of a compound and/or other biologically active agent within the methods and formulations of the disclosure is about 0.01 mg/kg body weight to about 10 mg/kg body weight, such as about 0.05 mg/kg to about 5 mg/kg body weight, or about 0.2 mg/kg to about 2 mg/kg body weight. Dosage can be varied by the attending clinician to maintain a desired concentration at a target site (for example, the lungs or systemic circulation). Higher or lower concentrations can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, or intranasal delivery versus intravenous or subcutaneous delivery. Dosage can also be adjusted based on the release rate of the administered formulation, for example, of an intrapulmonary spray versus powder, sustained release oral versus injected particulate or transdermal delivery formulations, and so forth. To achieve the same serum concentration level, for example, slow- release particles with a release rate of 5 nanomolar (under standard conditions) would be administered at about twice the dosage of particles with a release rate of 10 nanomolar.

The instant disclosure also includes kits, packages and multi-container units containing the herein described pharmaceutical compositions, active ingredients, and/or means for administering the same for use in the prevention and treatment of diseases and other conditions in mammalian subjects. Kits for diagnostic use are also provided. In one embodiment, these kits include a container or formulation that contains one or more of the conjugates described herein. In one example, this component is formulated in a pharmaceutical preparation for delivery to a subject. The conjugate is optionally contained in a bulk dispensing container or unit or multi-unit dosage form. Optional dispensing means can be provided, for example a pulmonary or intranasal spray applicator. Packaging materials optionally include a label or instruction indicating for what treatment purposes and/or in what manner the pharmaceutical agent packaged therewith can be used.

Example 1

Acetamide, 2-[4-[3-[(4-cyano-3-trifluoro-methyl)phenyl]-5,5-dimethyl-2,4- dioxoimidazol-idin- 1 -yl]-but-2-ynyloxy]-N-[(7S)-5,6,7,9-tetrahydro- 1,2,3,10-tetramethoxy-9- oxobenzo[a]heptalen-7-yl] (hereafter referred to as "CCN", for colchicine-cyanonilutamide) was synthesized as detailed below. CCN binds to tubulin and inhibits tubulin assembly with greater potency than colchicine and has activity that is comparable to the more potent thiocolchicine. Despite the hydrophobic binding pocket of the AR ligand-binding domain and the relatively bulky nature of colchicine, CCN still retains AR binding activity. The structural basis of binding to the AR ligand-binding domain was determined using molecular modeling techniques. The alkyne-based linker allows for a sufficient distance between the cyanonilutamide and colchicine moieties to allow the former to bind in the binding pocket and at the same time for the latter to extend outside AR. Furthermore, colchicine and CCN increase cytoplasmic AR protein levels in prostate cancer cells. Moreover, CCN is more potent in killing androgen independent prostate cancer cells than colchicine as well as the combination of colchicine and nilutamide.

Chemistry

¹H NMR spectra were recorded at 400, 500 or 600 MHz, and signals are given in ppm. J values are given in hertz. Mass spectra were determined by electrospray (ES). Column chromatography was carried out using Merck 60 silica gel (70-230 mesh). TLC was performed on Merck 60 F₂₅₄ silica gel glass plates. Elemental analyses were performed by Atlantic Microlabs (Norcross, GA). Melting points are uncorrected. Unless stated, all solvents and reagents were purchased from commercial sources and used without further purification.

Benzonitrile, 4-[3-(4-hydroxy-2-butynyl)-4,4-dimethyl-2,5-dioxo-l-imidazolidinyl]-2- (trifluoromethyl)- (2). To 1 (15) (3.90 g, 13.1 mmol) in DMF (60.0 mL) was added cesium carbonate (3.85 g, 1 1.8 mmol) and a solution of 4-chloro-2-butyn-l-ol (3.60 g, 34.4 mmol) in DMF (5.0 mL). After 3 hours at room temperature, the mixture was filtered. The filtrate was diluted with EtOAc (500 mL), washed with water (1.2 L), then dried (MgSO₄) and concentrated. The white solid was dried in vacuo to give 2 (2.85 g, 59%). ¹H NMR (500 MHz, DMSO-d₆): δ 8.32 (d, IH, J = 8.4 Hz); 8.20 (d, IH, J= 1.6 Hz); 8.06-8.04 (dd, 1 H, J= 1.7, 8.4 Hz); 5.16-5.14 (t, IH, J= 5.9 Hz); 4.30 (s, 2H); 4.08-4.07 (m, 2H); 1.54 (s, 6H). Mass spectroscopy: electrospray (positive ion): calc. for Ci₇H]₄F₃N₃O₃ = 365; found: m/z [relative intensity] 383 [(M+NH₄)⁺, 100%]. HPLC: Retention time 13.879 minutes with 100% peak area (Luna Cl 8, 4.6 x 250 mm, 5μ. Mobile phase: A=0.05M H₃PO₄ buffer, pH 3; B=acetonitrile + 1% water. Linear gradient= 95%A; 70%A; 100% B; 95%A.

Acetic acid, 2-[4-[3-[(4-cyano-3-trifluoromethyl)phenyl]-5,5-dimethyl-2,4- dioxoimidazolidin-l-yl]but-2-ynyloxy]-, methyl ester (3). A mixture of 2 (1.80 g, 4.93 mmol) and thallium ethoxide (349 μL, 4.93 mmol) in CH₃CN (17.0 mL) was stirred for 1 hour at ambient temperature then concentrated to dryness. The powder was dissolved in DMF (17.0 mL) and methyl bromoacetate (3.8 g, 24.8 mmol) was added. The mixture was heated at 6O⁰C for 3 hours, then cooled to room temperature and water (50 mL) was added. Extracted with Et₂O (4 x 100 mL), dried (MgSO₄) and concentrated. The crude product was purified by column chromatography (silica gel, hexanes/EtOAc, 1 :1) to give 3 (920 mg, 43%), m.p. 73- 75⁰C (uncorrected). Elemental analysis: calc: C(61.30), H(5.35), N(7.01); found: C(61.26), H(5.34), N(6.88). ¹H NMR (400 MHz, DMSOd₆): 5 8.33-8.31 (d, IH, J = 8.4 Hz); 8.19 (s, I H); 8.04-8.02 (dd, IH, J = 1.5, 8.0 Hz); 4.33 (s, 2H); 4.25 (s, 2H); 4.16 (s, 2H); 3.63 (s, 3H); 1.52 (s, 6H). Mass spectroscopy: electrospray (positive ion): calc. for C_2OHj₈F₃N₃O₅ = 437; found: m/z [relative intensity] 460 [(M+Na)⁺, 100%]. Thin layer chromatography (silica gel, E. Merck 60 F-254 glass plates): R_f value = 0.42 (EtOAc/hexanes, 1 :1).

Acetic acid, 2-[4-[3-[(4-cyano-3-trifluoromethyl)phenyl]-5,5-dimethyl-2,4- dioxoimidazolidin-l-yl]but-2-ynyloxy]- (4). A cold solution of 3 (500 mg, 1.14 mmol) and 1.0 M NaOH (1.178 mL, 1.178 mmol) in MeOH (7.50 mL) was stirred at room temperature for 2.5 hours. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (3 x 100 mL). The aqueous layer was acidified to pH 2 (1 N HCl) and extracted with CH₂Ch (4 x 100 mL). The combined organic layer was washed with water (3 x 100 mL) and brine (100 mL), then dried (Na₂SO₄) and concentrated to give 4 (231 mg, 48%). ¹H NMR (500 MHz,

DMSO-d₆): δ 12.69 (s, IH); 8.32-8.30 (d, IH, J= 8.4 Hz); 8.20 (d, IH₁ J = 1.3 Hz); 8.06-8.04 (dd, IH, J= 1.5, 8.4 Hz); 4.34 (s, 2H); 4.26 (s, 2H); 4.05 (s, 2H); 1.54 (s, 6H). Mass spectroscopy: electrospray (negative ion) calc. for Ci₉Hi₆F₃N₃O₅ = 423; found: m/z [relative intensity] 422 [(M-H)^', 100%].

Acetamide, 2-[4-[3-[(4-cyano-3-trifluoromethyl)phenyl]-5,5-dimethyl-2,4-dioxo-imidazolidin- l-yl]-but-2-ynyloxy]-N-[(7S)-5,6,7,9-tetrahydro-l,2,3,10-tetramethoxy-9- oxobenzo[a]heptalen-7-yl]- (6). To a solution of 5 (25) (175 mg, 0.490 mmol) and 4- methylmorpholine (176 μL, 1.60 mmol) in CHCI₃ (6.0 mL) was added a solution of 4 (226 mg, 0.534 mmol) in CHCl₃ (3.0 mL) and (benzotriazol-l-yloxy)tris-(dimethylamino)phosphonium hexafluorophosphate (BOP) (590 mg, 1.33 mmol). After 3.5 hours at room temperature, the reaction mixture was diluted with CHCl₃ (150 mL) and washed with sat. aq. citric acid (3 x 50 mL). The organic layer was concentrated to dryness, and the crude product was purified by column chromatography (silica gel, EtOAc/MeOH, 19:1) to give 6 (220 mg, 60%) as an orange solid. Elemental analysis: calc. for C₃₉H₃₇F₃N₄O₉ OJ hexanesO.6 H₂O: C(61.30), H(5.35),

N(7.01); found: C(61.26), H(5.34), N(6.88). ¹H NMR (600 MHz, DMSO-d₆): δ 8.59-8.57 (d, I H, J= 7.6 Hz); 8.32-8.31 (d, IH, J= 8.4 Hz); 8.20-8.19 (d, 1H, J= 1.3 Hz); 8.05-8.03 (dd, I H, J = 1.5, 8.4 Hz); 7.13 (s, IH); 7.1 1-7.01 (dd, 2H, J = 10.6, 50.2 Hz); 6.76 (s, IH); 4.41-4.37 (m, IH); 4.35 (s, 2H); 4.27 (s, 2H); 3.98-3.92 (dd, 2H, J= 14.9, 20.1 Hz); 3.86 (s, 3H); 3.83 (s, 3H); 3.78 (s, 3H); 3.52 (s, 3H); 2.60-2.56 (dd, IH, J= 6.0, 13.1 Hz); 2.23-2.18 (m, IH); 2.04-1.93 (m, 2H); 1.51 (s, 6H). Mass spectroscopy: electrospray (positive ion): calc. for C₃₉H₃₇F₃N₄O₉ = 762; found: m/z [relative intensity] 763 [(M+H)⁺,100%]. Thin layer chromatography (silica gel, E. Merck 60 F-254 glass plates): R_f value = 0.27 (EtOAc/MeOH, 19: 1).

Molecular modeling of CCN bound to AR Protein structure: Molecular modeling was based on the high-resolution structure (1.65A resolution) of human wild-type AR complexed with the agonist R-3 (PDB-code: 2AX9; (16)); a structure for apo-AR and AR with an antagonist bound has not yet been determined. The X-ray structures of rat AR with BMS-564929 (2NW4; (17)), a compound similar to cyanonilutamide, was resolved to only 3A. We aligned 2NW4 onto 2AX9. Based on the conformational differences in their ligand-binding site, the side chains of Asn705, Gln71 1 and Met895 in 2AX9 were modified allowing to accommodate cyanonilutamide. Missing loop residues 844-851 were added using the loopy vl .O ((18)) module from the Jackal 1.5 suite (Columbia University).

Preparation of molecular dynamics (MD) simulations: All MD simulations were performed with Gromacs 3.1 (19) using the Gromos 53 A6 force field. The ligands were built and prepared for MD simulations using Maestro (Schrδdinger, LLC) and PRODRG (20). A solvent box (including counterions) with at least IOA distance from box wall to any solute atom was added around the ligand-protein complex (approximately 37,500 atoms in total). All simulations were performed with a generalized reaction field and a dual group cutoff of 8A and 14A. Equilibration was performed using a NpT-ensemble, while steered MD simulations were run under NVT-conditions. A time step of 2fs was chosen using the LINCS algorithm for constraining bonds.

Steered MD: In steered MD (SMD), an in silico model for AFM experiments (21), a spring was attached to the ethyl group of cyanonilutamide ethylated at the 3-position of the 5,5-dimethyl- 2,4-dioxoimidazol-idin-l-yl portion. This ethyl group functions as starting elements of the linker and was initially oriented towards three channels. These paths were visually identified in the X-ray structure as possible channels (with similar dimension as the length of the linker) through which the hydrophobic linker could be topologically positioned when CCN binds to AR. The spring moves with constant velocity v along a predefined direction v. As the movement of the ligand along this direction experiences resistance, the spring is stretched, which results in external force F = k(vt - x) the ligand is experiencing. Simulations with different force constants k ranging from 50OkJ mol^'1 nm^'2 to 10,00OkJ mol^'1 nm^'2 and pulling velocities v ranging from 2nm ns^"1 to 5nm ns^"1 were performed. We finally chose k = 50OkJ mol^"1 nm^'2 and v = 3nm ns^"1, the same values as in a previous study on the thyroid receptor (1 1). This allows us to compare the resulting force profiles F(t) of ligands dissociating from two different species of the nuclear receptor family. For each channel the protein ligand complex was equilibrated for 1.5ns (0.5ns solvent equilibration only). Seven SMD simulations (Ins each) along each of the channels were run with slightly modified directions.

Equilibration of the AR-CCN complex: For channels 1 and II the linker was grown into the channel in three steps, always merging between two and four additional heavy atoms to cyanonilutamide. The resulting new structure was equilibrated using 2500 steps of steepest- descent energy minimization and Ins of MD simulations, before the linker was elongated any further. Finally, the colchicine moiety was attached to the cyanonilutamide-linker compound, and the full system was equilibrated using a 10ns MD simulation.

Tubulin assembly and tubulin binding studies

Inhibition of tubulin assembly (22) and inhibition of colchicine binding (23) were measured as described in detail previously. In the assembly reaction, 10 μM tubulin was used. In the colchicine binding reaction tubulin was at 1.0 μM and both [³H]colchicine and the inhibitor at 5 μM. Combretastatin A-4 was generously provided by Dr. G. R. Pettit of Arizona State University and thiocolchicine by Dr. A. Brossi of the National Institute of Diabetes and Digestive and Kidney Diseases. Colchicine was purchased from Sigma. Tritiated mibolerone (MIB) binding studies

The AR binding affinity of synthetic AR ligands was determined using an in vitro radioligand competitive binding assay, as previously described (24). Briefly, an aliquot of AR cytosol isolated from the ventral prostates of castrated male rats was incubated with 1 nM of [³H]MIB and 1 mM of triamcinolone acetonide at 4 ⁰C for 18 h in the absence or presence of ten increasing concentrations of CCN (10^"1 nM to 10⁴ nM). Nonspecific binding Of [³H]MIB was determined by adding excess unlabeled MIB (1000 nM) to the incubate in separate tubes. After incubation, the AR-bound radioactivity was isolated using the HAP method (24). The bound radioactivity was then extracted from HAP and counted. The specific binding of [³H]MIB at each concentration of the compound of interest was calculated by subtracting the nonspecific binding Of [³H]MIB, and expressed as the percentage of the specific binding in the absence of the compound of interest (BO). The concentration of CCN that reduced BO by 50% (i.e., IC₅₀) was determined using WinNonlin (Pharsight Corporation, Mountain View, CA). The equilibrium binding constant (K₁) of the compound of interest was calculated by K, = Kd IC₅₀ /(K_d + L), where Kd was the dissociation constant Of [³H]MIB (0.19 ± 0.01 nM), and L was the concentration of [³H]MIB used in the experiment (1 nM). The K₁ value of each compound of interested was further compared.

Cell Culture, Cell Survival Assay and Western Blots

LNCaP cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum and glutamine. Cells were exposed to vehicle, 0.1 μM nilutamide, 0.1 μM colchicine and 0.1 μM CCN for 11 hours and cells were washed with PBS. Protein was crosslinked in situ by treating with 1 mM dithiobis(succinimidyl)propionate (DSP) (Pierce, Rockford, IL) for 30 minutes at room temperature and gently rocked. Excess DSP was neutralized with IM Tris PH 7.5. Nuclear and cytoplasmic protein extracts were prepared using the Pierce nuclear and cytoplasmic fractionation kit. Forty μg protein was analyzed on a 4-20% Tris-glycine gel and protein was transferred to a polyvinylidene fluoride membrane, blocked in 1% fish gelatin in PBS, incubated sequentially with rabbit anti-AR antibody (Cell Signaling, Danvers, MA) and a horseradish peroxidase-labled anti-rabbit secondary antibody (Amersham, GE Healthcare, Waukesha, Wl) using standard methods. Membranes were then incubated with horseradish peroxidase substrate solution (Amersham) prior to exposure to film. LAPC4AI cells were obtained courtesy of Dr. Charles Sawyers and grown in Iscove's Modified Eagle media supplemented with 10% fetal bovine serum and glutamine. Cells were plated at 50,000 cells per well on a 96 well plate. The following day, 0.01, 0.05 and 0.1 μM colchicine and CCN were added to cells, in addition to the combination of 0.05 μM colchicine and 0.05 μM nilutamide, all in triplicate. After 48 hours of incubation, cell survival was measured with the CellTiter-Blue assay (Promega, Madison, WI), as specified by the manufacturer.

Results

Linking the C7 acetamido group of colchicine through an alkyne linker to cyanonilutamide results in CCN. Cyanonilutamide is a derivative of the AR antagonist nilutamide, which is in clinical use for prostate cancer. Combretastatin A-4 and thiocolchicine are structurally related to colchicine and also bind at the tubulin colchicine site. CCN was designed as a bifunctional compound that would interact with both tubulin and AR. All structures are shown in FIG. 1.

Compound 1 was prepared by the method of Cogan and Koch (15) (see FIG. 2). Alkylation of 1 with 4-chloro-2-butyn-l-ol gives alcohol 2. Alkylation of the thallium salt of 2, followed by hydrolysis gives carboxylic acid 4. Intermediate 5 was prepared in three steps using the method of Bagnato, Eilers, Horton and Grissom (25). BOP coupling of 4 and 5 gives the compound 6 (CCN) in 60% yield.

As a first step to evaluate CCN, we sought to determine AR binding activity. Tritiated mibolerone binding studies of CCN showed that the K₁ of this compound is 449 +/- 49 nM, and in the same experiments the K, of dihydrotestosterone is 0.2-0.3 nM. Hydroxyflutamide, which is the active metabolite of flutamide, a clinically used AR antagonist, has a K₁ of about 50 nM (26). Therefore, despite the bulky colchicine side chain, CCN binds AR with only a one log lower affinity than a clinically active AR antagonist. The AR binding activity of CCN suggests that the rigid alkyne linker functions as designed to extend the colchicine moiety outside the enclosed hydrophobic AR ligand-binding pocket with preservation of the AR-binding activity of cyanonilutamide.

To investigate the structural basis of CCN binding to AR, we employed molecular modeling techniques based on known X-ray structures of AR-ligand complexes. Visual inspection of the modified AR X-ray structure (see Materials and Methods) with a compound structurally similar to cyanonilutamide (BMS-564929) revealed three small channels of appropriate length for the linker to extend the colchicine moiety outside of the AR ligand binding domain. Channel I is composed of the region between helices 3,6,7 and 1 1 , channel II between helices 1 1 and 12 and channel III the mobile region between helices 1 and 2 as well as helices 3 and 5. Topological feasiblity of a channel to accommodate the linker region of CCN is a necessary, but not sufficient, property for binding. The channel must also allow the cyanonilutamide portion of the compound to enter and exit the hydrophobic binding pocket of AR. To address this issue, we performed SMD simulations of cyanonilutamide ethylated at the 3-position of the 5,5-dimethyl-2,4-dioxoimidazol-idin-l-yl portion, pulling the ethyl group along the three channels (see Table 1 below).

Table 1. Maximum and integrated force of the average force profiles.

Although SMD along channel I yields the highest maximum force, none of the paths is statistically favored over the others, taking the standard deviation of about 30-50 pN over seven simulations along each path into account. Considering the integrated force, i.e., the total work performed (with a standard deviation of about 20 pN ns), path I and path II are energetically favored over path III, and these values are lower than comparable values for the thyroid receptor (1 1). Consequently, CCN was docked in two different binding modes, with its linker bound within channel I or II.

Standard MD equilibration simulations of CCN with its linker bound within channel I (FIG. 3), showed that this configuration of binding occurs without substantial conformational changes in the protein. The length and hydrophobic character of the channel is perfectly suited for the predominantly hydrophobic linker, such that cyanonilutamide binds in the binding pocket of AR and the colchicine moiety resides outside the AR ligand-binding domain. After 10 ns equilibration, a conformational change in helix 12 and the C-terminus of helix 1 1 was observed, while the other portions of helices comprising channel I remained almost unaltered. One can only speculate if the observed change in helix 11 , and especially in helix 12, might reflect an antagonistic effect on AR (similar to conformational changes observed in estrogen receptor (27), for example); no experimental X-ray structure of wildtype AR with an antagonist has yet been determined yet.

MD simulations of CCN binding to AR, with the linker positioned in channel II (FIG. 4), results in a significant conformational change in helix 11. However, we should mention that according to the proposed mouse-trap model (28) for ligand association with nuclear receptors, the conformations of helices 1 1 and 12 might differ from the X-ray structures used as starting points for our simulations. Due to lack of experimental structural information of apo or antagonist structures for AR, these scenarios cannot be ruled out.

There are extensive structure activity relationship data for colchicine in the literature (13). Further, the colchicine binding site on tubulin has been defined by X-ray crystallography (29). Although the colchicine binding site is primarily in the β-subunit of tubulin, the drug binds close to the intradimer α-β interface (29). Thus, colchicine analogs also interact with the tubulin α-subunit (13). Colchicine only binds to the soluble tubulin heterodimer and induces a conformational change, thereby inhibiting tubulin polymerization (13). We found that CCN inhibited tubulin assembly with an IC₅₀ of 1.1 +/- 0.1 μM and is a better inhibitor of tubulin assembly than colchicine itself (see Table 2 below).

Table 2. Inhibitory effects of CCN, colchicine, thiocolchicine, and combretastatin A-4 on tubulin assembly and on binding of [³H]colchicine to tubulin.

CCN was also examined for its ability to inhibit the binding of [³H]colchicine to tubulin, in comparison with thiocolchicine and combretastatin A-4. Inhibitory effects on tubulin assembly and on colchicine binding were equivalent to those of thiocolchicine (Table 2). In comparison with combretastatin A-4, CCN was a more effective inhibitor of assembly, but it was less potent as an inhibitor of [³H]colchicine binding. This potent inhibition of colchicine binding by combretastatin A-4 derives entirely from its rapid binding to tubulin, in comparison with the slower binding of colchicinoids (30). Given the increased activity of CCN over colchicine and the fact that some steroid receptor ligands have tubulin binding activity (31), it was verified that cyanonilutamide had no effect on tubulin assembly (data not shown).

The affect of CCN on AR localization was then examined. LNCaP prostate cancer cells have a mutation in the AR ligand-binding domain, which changes the antagonist activity of some clinically used prostate cancer drugs to agonist activity (32). This sort of change may account for the anti-androgen withdrawal phenomenon seen in some patients (33). Therefore, any effect of CCN on AR cellular localization, though it may be related to AR binding, should not be related to classical antagonist activity. LNCaP cells have both nuclear and cytoplasmic AR without stimulation. It was found that treatment with 0.1 μM CCN or 0.1 μM colchicine increased cytoplasmic AR protein levels without a detectable change in nuclear AR (FIG. 6). Treatment with 0.1 μM nilutamide decreased cytoplasmic AR and increased nuclear AR, as would be expected with nuclear import. The increase in cytoplasmic AR upon treatment with CCN or colchicine is most likely related to tubulin depolymerization and the inability of dyenin- and microtubule-dependent steroid receptor nuclear import to occur.

Next, the effect of CCN on the growth of the androgen independent LAPC4AI cell line was tested. Cells were treated with CCN, colchicine and a combination of colchicine and nilutamide for 48 hours (FIG. 7). It was found that CCN inhibits androgen independent cell survival more than colchicine, with a difference that is maximal at 0.05 μM. To rule out the possibility that the cell toxicity of CCN is reproducible with a combination of colchicine and an AR antagonist, LAPC4AI cells were also treated with a combination of 0.05 μM colchicine and 0.05 μM nilutamide. This combination resulted in toxicity that is the same as 0.05 μM colchicine alone. Example 2

Compounds with taxane moieties were made according to the synthesis described in detail below. The first compound prepared was VTl 92-183. In this construct the AR ligand was linked to the C7 position of paclitaxel. The second compound, VTl 92-204, was related to the first, but the linkage between the AR ligand and paclitaxel was lengthened by incorporation of a succinic acid group in place of the carbonate group of VT192-183. The third compound, VT 192-210, was similar to the second compound, differing only by having a glutaric acid in place of the succinic acid moiety. The fourth compound (VT 192-219) was a derivative of docetaxel, and this required the synthesis of docetaxel from 10-deacetylbaccatin III. T-

(Triisopropylsilyl)-7-(triethylsilyl)-10-acetyldocetaxel was first prepared, and this was then converted to 2'-(triisopropylsilyl)-7-(triethylsilyl)docetaxel and thence to VT 192-219. These conversions required selective deprotection at ClO, followed by acylation at ClO with succinic anhydride, followed by coupling with the AR ligand and deprotection. Additional compounds were prepared with various longer linkers to optimize AR binding, and with polyethylene glycol (PEG) linkers to increase aqueous solubility. The first of these incorporates a polyethylene glycol (PEG) linker between docetaxel and the AR ligand. The AR ligand was first prepared with two different PEG linkers attached to the alkyne unit, and these modified AR ligands were then coupled with 2'-(triisopropylsilyl)-7- (triethylsilyl)docetaxel to give VT192-297 and VT192-300. Another compound (VT206-21) was prepared by coupling the AR-TEG ligand to the 7-position of docetaxel through a succinyl linker. Due to the promising activity of compound VTl 92-219, the related docetaxel derivative VT206-85 was synthesized, whose linker is one methylene longer than VTl 92-219. The synthesis of VT206-85 began with a previous intermediate 2'-(triisopropylsilyl)-7- (triethylsilyO-lO-deacetyldocetaxel, which was treated with glutaric anhydride and DMAP, and was finally converted to VT206-85. Also synthesized was a docetaxel analog with the androgen receptor ligand linked to other positions than position 10. Compound VT206-92 has the AR ligand cyanonilutamide-alkyne joined to docetaxel at the 3'-N position through succinic anhydride. Two more docetaxel derivatives, VT206-105 and VT 206-106, were synthesized. They both had the AR ligand connected to docetaxel with longer PEG linkers than the previous compounds VT 192-297 and VT 192-300. The detailed synthesis appears below:

To the solution of cyanonilutamide (825 mg, 2.78 mmol) in DMF (8.5 mL) was added NaH (60% in mineral oil, 134 mg, 3.34 mmol) under argon at room temperature. After stirring for 15 minutes at ambient temperature, the mixture was treated dropwise with a solution of tert- butyl(4-iodobut-2-ynyloxy)dimethylsilane (1.01 g, 3.34 mmol) in anhydrous DMF (8.5 mL). The mixture was stirred at room temperature for 3 hours. Then the solution was poured into saturated brine and extracted with ethyl acetate (3X 100 mL). The combined organic extracts were washed with water and saturated brine, and dried over Na₂SO₄. After vacuum filtration, the solution was rotary evaporated and the residue was purified by flash chromatography (30% ethyl acetate in hexane) to give TBS ether 1 (1.05 g, 81.5%) as a white solid.

To TBS ether 1 (885 mg, 1.88 mmol) was added the solution of HCl in ethanol (1%, 60 mL) at room temperature. After stirring for 15 minutes, the mixture was treated with saturated NaHCO₃ solution carefully and extracted with ethyl acetate (3X 150 mL). The combined organic phase was then washed with saturated brine, dried over NaSO₄, vacuum filtered, and rotary evaporated. The residue was purified by flash chromatography (50% ethyl acetate in hexane) to afford a white solid 2 (543 mg, 81.4%).

[1] Cogan, P S and Koch, T H , Rational Design and Synthesis of Androgen Receptor-Targeted Nonsteroidal Anti-Androgen Ligands for the Tumor-Specific Delivery of a Doxorubicin-Formaldehyde Conjugate, J Med Chem , 2003, 46, 5258-5270 To the solution of compound 2 (61 mg, 0.167 mmol) in DCM (1.6 mL) was added succinic anhydride (50 mg, 0.5 mmol) and DMAP (62 mg, 0.5 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. After vacuum filtration and rotary evaporation, the residue was then purified by flash chromatography (70% ethyl acetate in hexane) to afford 3 as a white solid (70 mg, 90%).

To solution of paclitaxel (55.4 mg, 0.065 mmol) in DMF (0.5 mL) was added imidazole (22.1 mg, 0.325 mmol) and TBSCl (98 mg, 0.65 mmol) successively at room temperature. The mixture was stirred overnight at 60 ⁰C, then treated with saturated NaHCO₃ solution, and extracted with ethyl acetate (3X 10 mL). The combined organic layers were washed with water and saturated brine, dried over NaSO₄, vacuum filtered, and concentrated. The resulting residue was purified by flash chromatography (50% ethyl acetate in hexane) to give a white solid 4 (58 mg, 92.3%).

JQ192-183

Bis(4-nitrophenyl) carbonate (20 mg, 0.063 mmol) and DMAP (1 1 mg, 0.090 mmol) was added to the solution of compound 4 (20 mg, 0.21 mmol) in DCM (0.1 mL). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 10 mL), washed with saturated brine, and dried over NaSO₄. The extract was then vacuum filtered and concentrated. The residue was purified by flash chromatography (30% ethyl acetate in hexane) to give a white solid (16 mg, 67%).

Then this white solid (16 mg, 0.014 mmol), compound 2 (15 mg, 0.042 mmol) and DMAP (5 mg, 0.042 mmol) was dissolved in DCM (0.1 mL). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 10 mL), washed with saturated brine, and dried over NaSO₄, followed by vacuum filtration and rotary evaporation. The residue was then purified by flash chromatography (50% ethyl acetate in hexane) to afford a white solid (15.7 mg, 82%).

To the solution of the resulting white solid (15.7 mg, 0.012 mmol) in THF (1 mL) was added HF-pyridine (0.2 mL) at 0 ⁰C. The mixture was stirred at room temperature and the reaction was monitored by TLC. When the reaction completed, saturated NaHCOa solution was added to the reaction mixture. The mixture was then extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. After vacuum filtration and rotary evaporation, the residue was then purified by flash chromatography (50% ethyl acetate in hexane) to afford compound JQ192-183 (also referred to herein as "VTl 92-183") (12.9 mg, 86.4%) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 8.13 (d, 1 H, J = 2.0 Hz), 8.1 1 (dd, 2H, J = 8.0, 1.5Hz), 7.99 (dd, IH, J = 8.5, 2.0 Hz), 7.92 (d, IH, J = 8.5 Hz), 7.75 (dd, 2H, J = 8.5, 1.5 Hz), 7.62 (tt, 1H, J= 7.5, 1.5 Hz), 7.52-7.40 (m, 9H), 7.35 (tt, 1H, J= 7.5, 1.5 Hz), 7.01 (d, IH, J= 9.0 Hz), 6.28 (s, IH), 6.18 (t, IH, J= 9.0 Hz), 5.79 (dd, IH₁ J= 9.0, 2.5 Hz), 5.66 (d, IH, J = 7.0 Hz), 5.46 (dd, IH₁ J= 9.0, 2.5 Hz), 4.93 (d, IH₁ J= 7.0 Hz)₁ 4.91 (dt, IH₁ J= 16.0, 2.0 Hz), 4.79 (dd, IH, J= 4.0, 2.5 Hz), 4.67 (dt, IH, J= 16.0, 2.0 Hz), 4.32 (d, I H, J= 8.0 Hz), 4.28 (t, 2H₁ J= 2.0 Hz)₁ 4.17 (d, IH₁ J= 8.5 Hz), 3.91 (d, IH₁ J= 6.5 Hz)₁ 3.61 (broad s, I H)₁ 2.58 (ddd, IH₁ J= 15.0, 10.0, 7.5 Hz)₁ 2.38 (s, 3H), 2.33 (d, IH₁ J= 9.5 Hz)₁ 2.32 (d, IH₁ J = 9.0 Hz)₁ 2.15 (s, 3H)₁ 1.97 (ddd, IH₁ J= 14.5, 10.5, 2.0 Hz), 1.84 (d, 3H, J= 1.0 Hz), 1.80 (s, 3H), 1.71 (broad s, IH)₁ 1.61 (s, 6H), 1.21 (s, 3H), 1.16 (s, 3H); ¹³C NMR (500 MHz, CDCl₃) δ 201.6, 174.4, 172.7, 170.6, 169.2, 167.1 , 166.9, 153.7, 152.4, 140.6, 138.0, 136.3, 135.4, 133.9, 133.7, 133.0, 132.1, 130.3, 129.1, 128.9, 128.8, 128.5, 128.1, 127.2, 123.2, 1 15.1, 108.6, 83.8, 81.7, 81.0, 78.6, 78.3, 76.5, 76.1, 75.4, 74.3, 72.3, 62.2, 56.3, 55.9, 55.0, 47.0, 43.3, 35.6, 33.5, 29.2, 26.6, 23.3, 22.6, 21.00, 20.9, 14.7, 10.7; MS m/z 1245.4193 (M⁺+l).

To paclitaxel analog 4 (18.7 mg, 0.02 mmol) in DCM (0.2 mL) was added succinic anhydride (7.1 mg, 0.07 mmol) and DMAP (7.8 mg, 0.06 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 10 mL), washed with saturated brine, dried over NaSO₄, vacuum filtered and rotary evaporated. The residue was then purified by flash chromatography (60% ethyl acetate in hexane) to afford a white solid 5 (21.4 mg, 100%).

To the solution of white solid 5 (21.4 mg, 0.02 mmol), compound 2 (21.4 mg, 0.06 mmol) and DMAP (7 mg, 0.06 mmol) in DCM (1 mL) was added EDAC (1 1.5 mg, 0.06 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. Then the mixture was vacuum filtered and rotary evaporated. Without purification, the residue (19.4 mg) was dissolved in THF (1 mL) and HF-pyridine (0.23 mL) was added at 0 ⁰C. The mixture was stirred at room temperature until the completion of the reaction (monitored by TLC). Then saturated NaHCO₃ solution was added to the reaction mixture. The mixture was then extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. After vacuum filtration and rotary evaporation, the residue was then purified by flash chromatography (50% ethyl acetate in hexane) to give compound JQl 92-204 (also referred to herein as "VTl 92-204") (14 mg, 53.8% for two steps) as a white solid: ¹H NMR (500 MHz,

CDCl₃) δ 8.12 (d, IH, J= 2.0 Hz), 8.10 (dd, 2H, J = 8.0, 1.5Hz), 7.97 (dd, IH, J = 8.0, 2.0 Hz), 7.89 (d, IH, 7= 8.0 Hz), 7.76 (dd, 2H, J= 8.5, 1.5 Hz), 7.62 (tt, IH, /= 7.5, 1.5 Hz), 7.52-7.40 (m, 9H), 7.34 (tt, IH, J= 7.0, 1.5 Hz), 7.04 (d, IH, J= 9.0 Hz), 6.16 (s, IH), 6.16 (t, IH, J= 9.0 Hz), 5.79 (dd, IH, J= 9.0, 2.5 Hz), 5.66 (d, IH, J= 7.0 Hz), 5.56 (dd, IH, J= 10.8, 7.0 Hz), 4.92 (d, I H, J= 10.0 Hz), 4.79 (d, I H, J= 3.0 Hz), 4.67 (d, 2H, J= 3.5 Hz), 4.30 (d, IH, J= 8.5 Hz), 4.27 (d, 2H, J= 2.0 Hz), 4.17 (d, I H, J = 8.5 Hz), 3.90 (d, I H, J= 7.0 Hz), 3.61 (d, 1 H, J = 5.0 Hz), 2.69 (m, 1 H), 2.66-2.54 (m, 4H), 2.36 (s, 3H), 2.33 (d, 1 H, J = 9.0 Hz), 2.32(d, 1 H, J = 9.0 Hz), 2.15 (s, 3H), 1.83 (m, I H), 1.81 (s, 3H), 1.80 (s, 3H), 1.61 (d, 6H, J= 2.5 Hz), 1.20 (s, 3H), 1.13 (s, 3H); ¹³C NMR (500 MHz, CDCl₃) δ 202.0, 174.4, 172.6, 171.9, 171.3, 170.6, 169.2, 167.0, 152.5, 140.6, 138.2, 136.4, 135.5, 133.9, 133.8, 133.0, 132.1, 130.4 129.1, 128.9, 128.7, 128.3, 127.3, 123.1, 1 15.0, 108.6, 83.9, 81.2, 80.9, 78.9, 78.7, 76.6, 75.5, 74.4, 73.3, 72.2, 71.9, 62.3, 56.2, 55.0, 52.1, 47.1, 43.5, 35.8, 33.5, 29.2, 29.1, 28.8, 26.7, 23.2, 22.6, 20.9, 14.8, 10.9; MS m/z 1301.4403 (M⁺+l).

To paclitaxel analog 4 (18 mg, 0.019 mmol) in DCM (1 mL) was added glutaric anhydride (6.5 mg, 0.057 mmol) and DMAP (7 mg, 0.057 mmol). The mixture was stirred at room temperature for 2 days. After the addition of water, the mixture was extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄, followed by vacuum filtration and rotary evaporation. The residue was then purified by flash chromatography (60% ethyl acetate in hexane) to afford a white solid (10 mg, 48.7%).

Then to the solution of this white solid (10 mg, 0.01 mmol), compound 2 (1 1 mg, 0.03 mmol) and DMAP (4 mg, 0.03 mmol) in DCM (1 mL) was added EDAC (6 mg, 0.03 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. Then the mixture was vacuum filtered, concentrated and vacuum dried. Without purification, the residue (12.3 mg) was dissolved in THF (1 mL) and HF-pyridine (0.2 mL) was added at 0 ⁰C. The mixture was stirred at room temperature and the reaction was monitored by TLC. When starting material was completely consumed, saturated NaHCO₃ solution was added to the reaction mixture. The mixture was then extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. After vacuum filtration and rotary evaporation, the residue was then purified by flash chromatography (50% ethyl acetate in hexane) to give compound JQ192-210 (also referred to herein as "VT192-210") (10.2 mg, 77.6% for two steps) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 8.13 (d, 1 H, J = 2.0 Hz), 8.10 (d, 2H, J = 8.0 Hz), 7.99 (dd, I H, J= 8.5, 2.0 Hz), 7.92 (d, IH, J= 8.5 Hz), 7.76 (d, 2H, J = 8.0 Hz), 7.62 (t, IH, J= 7.5 Hz), 1.52-1 Al (m, 5H), 7.43-7.39 (m, 4H), 7.34 (t, IH, J= 7.0 Hz), 7.05 (d, I H, J = 8.5 Hz), 6.19 (s, IH), 6.16 (t, IH, J= 9.0 Hz), 5.79 (dd, I H₁ J= 9.0, 2.0 Hz), 5.65 (d, IH₅ J = 6.0 Hz), 5.54 (dd, I H, J= 10.0, 8.0 Hz), 4.92 (d, IH, J= 8.5 Hz), 4.79 (d, IH, J= 3.0 Hz), 4.66 (s, 2H), 4.31 (d, I H, J= 8.5 Hz), 4.28 (s, 2H), 4.17 (d, I H, J= 8.5 Hz), 3.90 (d, I H, J= 7.5 Hz), 3.61 (d, I H, J= 5.0 Hz), 2.57 (ddd, I H₁ J= 14.5, 9.5, 8.0 Hz)₁ 2.45-2.36 (m, 4H)₁ 2.37 (s, 3H), 2.32 (s, I H)₁ 2.31 (s, IH)₁ 2.14 (s, 3H), 1.91 (m, 2H)₁ 1.82 (m, I H)₁ 1.82 (s, 3H)₁ 1.79 (s, 3H), 1.61 (s, 6H)₁ 1.19 (s, 3H)₁ 1.15 (s, 3H); ¹³C NMR (500 MHz₁ CDCl₃) δ 202.0, 174.4, 172.5,

172.3, 172.0, 170.6, 169.0, 167.1, 152.5, 140.7, 138.2, 136.4, 135.5, 133.9, 133.8, 133.0, 132.0, 130.3, 129.2, 128.9, 128.5, 128.1, 127.2, 123.2, 115.1, 108.6, 84.0, 81.1, 80.9, 78.8, 78.6, 76.6, 75.4, 74.4, 73.3, 72.2, 71.6, 62.2, 56.3, 55.0, 52.0, 47.0, 43.3, 35.6, 33.6, 33.1, 29.3, 26.6, 23.3, 22.7, 20.9, 19.6, 14.8, 10.9; MS m/z 1315.4564 (M⁺+ 1).

To the solution of docetaxel analog 6² (310 mg, 0.277 mmol) in ethanol (20 mL) was added hydrazine monohydrate (4.8 mL). The mixture was stirred at room temperature overnight. After the addition of saturated NH₄Cl solution, the mixture was extracted with ethyl acetate (3X 100 mL), washed with saturated brine, and dried over NaSO₄, followed by vacuum filtration and rotary evaporation. The residue was then purified by flash chromatography (15% ethyl acetate in hexane) to afford white solid 7 (253 mg, 84.8%).

To the solution of docetaxel analog 7 (36 mg, 0.033 mmol) in toluene (2 mL) was added succinic anhydride (15 mg, 0.15 mmol) and DMAP (13 mg, 0.1 mmol). The mixture was stirred at 85-9O ⁰C for 24 hours. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate and aqueous HCl solution (0.2%) was added carefully. The aqueous layer was then extracted with ethyl acetate (3X 30 mL). The combined organic phase was washed with saturated brine and dried over NaSO₄. After vacuum filtration and rotary evaporation, the residue was purified by flash chromatography (60% ethyl acetate in hexane) to afford white solid 8 (16 mg, 41%).

JQ182-21S

[2] Ojima I , Fumero-Oderda, C L , Kuduk, S D , Ma, Z , Kiπkae, F And Kiπkae, T , Structure-Activity Relationships Study of Taxoids for Their Ability to Activate Murine Macrophages as well as Inhibit the Growth of Macrophage-like Cells, Bioυrg Med Chem , 2003, 11 , 2867- 2888 To the solution of docetaxel analog 8 (22 mg, 0.019 mmol), compound 2 (22.3 mg, 0.057 mmol) and DMAP (7 mg, 0.057 mmol) in DCM (1 mL) was added EDAC (1 1 mg, 0.057 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. Then the mixture was vacuum filtered and rotary evaporated. Without purification, the residue (28 mg) was dissolved in THF (1 mL) and HF-pyridine (0.3 mL) was added at 0 ⁰C. The mixture was stirred at room temperature until the completion of the reaction (monitored by TLC). Then saturated NaHCO₃ solution was added to the reaction mixture. The mixture was extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. After vacuum filtration and rotary evaporation, the residue was then purified by flash chromatography (50% ethyl acetate in hexane) to give compound JQ192-219 (also referred to herein as "VT192-219") (13.3 mg, 55.8% for two steps) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 8.13 (d, IH, J= 2.0 Hz), 8.10 (d, 2H, J = 7.5 Hz), 7.99 (dd, IH, J = 8.0, 2.0 Hz), 7.90 (d, IH, J= 8.0 Hz), 7.61 (tt, IH, J= 7.5, 1.0 Hz), 7.50 (t, 2H, J = 8.0 Hz), 7.42-7.35 (m, 4H), 7.32 (tt, IH, J= 7.0, 1.0 Hz), 6.29 (s, IH), 6.22 (t, IH₁ J= 8.5 Hz), 5.65 (d, IH, J= 7.0 Hz), 5.35 (d, I H, J= 9.5 Hz), 5.25 (d, IH, J= 8.0 Hz), 4.93 (dd, IH, J= 9.0, 1.5 Hz), 4.71 (s, 2H), 4.61 (d, IH, J= 1.0 Hz), 4.38 (dd, IH, J= 1 1.0, 6.5 Hz), 4.29 (d, IH, J= 8.0 Hz), 4.29 (s, 2H), 4.15 (d, I H, J= 8.0 Hz), 3.78 (d, IH, J= 7.0 Hz), 3.32 (s, IH), 2.91-2.83 (m, 2H), 2.83-2.66 (m, 2H), 2.51 (ddd, IH₅ J= 15.5, 10.0, 7.0 Hz), 2.43 (broad s, IH), 2.37 (s, 3H), 2.28 (m, 2H), 1.85 (m, IH), 1.84 (s, 3H), 1.66 (s, 3H), 1.62 (s, 6H), 1.33 (s, 9H), 1.25 (s, 3H), 1.13 (s, 3H); ¹³C

NMR (500 MHz, CDCl₃) δ 203.5, 174.5, 172.4, 171.3, 170.2, 167.2, 155.5, 152.4, 142.6, 138.3, 136.3, 135.4, 133.9, 132.9, 130.3, 129.1, 129.0, 128.8, 128.2, 128.1, 126.8, 123.2, 1 15.0, 108.8, 84.5, 81.2, 80.4, 79.2, 78.6, 76.5, 76.0, 75.0, 73.9, 72.5, 72.3, 62.3, 58.8, 56.3, 52.5, 45.7, 43.3, 35.8, 35.6, 29.2, 29.0, 28.9, 28.3, 26.9, 23.3, 22.8, 22.1, 15.0, 9.7; MS m/z 1255.4573 (M⁺+l ).

Di(ethylene glycol) (1 mL, 10.51 mmol) and pyridine (0.85 mL, 10.51 mmol) were dissolved in DCM (8 mL) and the solution of TBSCl (1.58 g, 10.51 mmol) in DCM (4 mL) was added at 0⁰C. The mixture was stirred at room temperature overnight. The reaction solution was concentrated and purified by flash chromatography (50% ethyl acetate in hexane) to give a colorless oil (900 mg, 38.9%).

To the solution of this colorless oil (44 mg, 0.2 mmol), compound 3 (31 mg, 0.067 mmol) and DMAP (25 mg, 0.2 mmol) in DCM (4 mL) was added EDAC (40 mg, 0.2 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 20 mL), washed with saturated brine, and dried over NaSO₄. Then the mixture was vacuum filtered and rotary evaporated. Without purification, the residue (25 mg) was dissolved in the solution of HCl in ethanol (1%, 1.2 mL) at room temperature. After stirring for 15 minutes, the mixture was treated with saturated NaHCO₃ solution carefully and extracted with ethyl acetate (3X 15 mL). The combined organic phase was then washed with saturated brine, dried over NaSO₄, vacuum filtered, and rotary evaporated. The residue was purified by flash chromatography (50% ethyl acetate in hexane) to afford compound 9 (19 mg, 51.3% for two steps) as a white solid.

To the solution of docetaxel analog 8 (20 mg, 0.017 mmol), compound 9 (1 1 mg, 0.020 mmol) and DMAP (6.7 mg, 0.051 mmol) in DCM (0.5 mL) was added EDAC (10 mg, 0.051 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 10 mL), washed with saturated brine, and dried over NaSO₄. Then the mixture was vacuum filtered and rotary evaporated. After purified by flash chromatography (40% ethyl acetate in hexane), the product (14.5 mg, 0.0085 mmol) was dissolved in pyridine (0.5 mL) and CH₃CN (0.25 mL). HF-pyridine (0.063 mL) was added to the solution at room temperature. The mixture was stirred at 62 ⁰C until the completion of the reaction (monitored by TLC). Then saturated NaHCO₃ solution was added to the reaction mixture dropwise. The mixture was extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. After vacuum filtration and rotary evaporation, the residue was then purified by flash chromatography (70% ethyl acetate in hexane) to give compound JQ192-297 (also referred to herein as "VTl 92-297") (10.2 mg, 41.6% for two steps) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 8.13 (d, IH, J = 1.5 Hz), 8.10 (d, 2H, J = 7.5 Hz), 7.99 (dd, I H₅ J= 8.5, 2.0 Hz), 7.91 (d, IH, J = 8.5 Hz), 7.61 (tt, 1H, J= 7.5, 1.5 Hz), 7.49 (t, 2H, J = 7.5 Hz), 7.42-7.35 (m, 4H), 7.32 (tt, I H, J = 7.0, 1.5 Hz), 6.30 (s, I H), 6.22 (t, IH, J = 8.5 Hz), 5.65 (d, IH, J= 7.0 Hz), 5.36 (d, I H, J = 10.0 Hz), 5.25 (d, I H, J= 9.0 Hz), 4.93 (dd, IH, J= 9.5, 2.0 Hz), 4.69 (t, 2H, J= 2.0 Hz), 4.61 (m, IH), 4.39 (dd, IH, J= 1 1.0, 7.0 Hz), 4.29 (d, IH, J= 8.5 Hz), 4.27 (t, 2H, J= 2.5 Hz), 4.27-4.23 (m, 4H), 4.16 (d, IH, J= 8.5 Hz), 3.78 (d, IH, J= 7.5 Hz), 3.71-3.67 (m, 4H), 3.34 (broad s, IH), 2.85 (dt, 2H, J= 6.5, 2.5 Hz), 2.81- 2.68 (m, 2H), 2.68-2.65 (m, 4H), 2.53 (ddd, 1H, J= 15.0, 9.5, 6.5 Hz), 2.47 (broad s, IH), 2.37 (s, 3H), 2.28 (m, 2H), 1.85 (m, IH), 1.84 (s, 3H), 1.72 (broad s, IH), 1.66 (s, 3H), 1.61 (s, 6H), 1.33 (s, 9H), 1.25 (s, 3H), 1.13 (s, 3H); ¹³C NMR (500 MHz, CDCl₃) δ 203.6, 174.4, 173.1, 172.5, 172.2, 172.0, 171.6, 170.3, 167.1, 155.5, 152.4, 142.7, 138.4, 136.3, 135.4, 133.8, 133.0, 130.3, 129.2, 129.0, 128.8, 128.2, 128.1, 126.8, 123.2, 1 15.0, 108.6, 84.5, 81.2, 81.1 , 80.4, 79.1, 78.6, 76.5, 75.9, 75.0, 73.7, 72.5, 72.2, 69.0, 63.9, 62.2, 58.6, 56.3, 52.3, 45.7, 43.3, 35.8, 35.6, 29.2, 29.1, 29.0, 28.9, 28.8, 28.2, 26.8, 23.2, 22.7, 22.0, 14.9, 9.6; MS m/z 1443.5204 (M⁺+l ).

Tri(ethylene glycol) (1 mL, 7.5 mmol) and pyridine (0.61 mL, 7.5 mmol) were dissolved in

DCM (6 mL) and the solution of TBSCl (1.1 g, 7.3 mmol) in DCM (3 mL) was added at O⁰C. The mixture was stirred at room temperature overnight. The reaction solution was concentrated and purified by flash chromatography (50% ethyl acetate in hexane) to give a colorless oil (800 mg, 41.5%). To the solution of this colorless oil (50 mg, 0.189 mmol), compound 3 (28 mg, 0.061 mmol) and DMAP (25 mg, 0.2 mmol) in DCM (4 mL) was added EDAC (40 mg, 0.2 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 20 mL), washed with saturated brine, and dried over NaSO₄. Then the mixture was vacuum filtered and rotary evaporated. Without purification, the residue (20 mg) was dissolved in the solution of HCl in ethanol (1%, 0.9 mL) at room temperature. After stirring for 15 minutes, the mixture was treated with saturated NaHCO₃ solution carefully and extracted with ethyl acetate (3X 15 mL). The combined organic phase was then washed with saturated brine, dried over NaSO₄, vacuum filtered, and rotary evaporated. The residue was purified by flash chromatography (60% ethyl acetate in hexane) to afford compound 10 (1 1 mg, 30.2% for two steps) as a white solid.

To the solution of docetaxel analog 8 (20 mg, 0.017 mmol), compound 10 (15 mg, 0.026 mmol) and DMAP (6.2 mg, 0.051 mmol) in DCM (0.8 mL) was added EDAC (10 mg, 0.051 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. Then the mixture was vacuum filtered and rotary evaporated. After purified by flash chromatography (50% ethyl acetate in hexane), the product (12 mg, 0.007 mmol) was dissolved in pyridine (0.4 mL) and CH₃CN (0.2 mL). HF-pyridine (0.052 mL) was added to the solution at room temperature. The mixture was stirred at 62 ⁰C until the completion of the reaction (monitored by TLC). Then saturated NaHCO₃ solution was added to the reaction mixture dropwise. The mixture was extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. After vacuum filtration and rotary evaporation, the residue was then purified by flash chromatography (70% ethyl acetate in hexane) to give compound JQ192-300 (also referred to herein as "VTl 92-300") (8.2 mg, 32.5% for two steps) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 8.13 (d, 1 H, J = 2.0 Hz), 8.10 (d, 2H, J = 7.0 Hz), 7.99 (dd, I H, J= 8.5, 2.0 Hz), 7.91 (d, IH, J = 8.5 Hz), 7.61 (tt, I H, 7= 7.0, 1.5 Hz), 7.49 (t, 2H, J = 7.5 Hz), 7.42-7.35 (m, 4H), 7.32 (tt, I H, J= 7.0, 1.5 Hz), 6.29 (s, I H), 6.22 (t, IH, J = 9.0 Hz), 5.65 (d, IH, J= 7.0 Hz), 5.37 (d, IH, J= 9.0 Hz), 5.25 (d, IH, J= 8.5 Hz), 4.93 (dd, 1 H, J= 9.O, 1.5 Hz), 4.68 (t, 2H, J= 2.0 Hz), 4.61 (m, IH), 4.38 (dd, I H, J=4.5, 3.5 Hz), 4.29 (d, IH, J= 8.5 Hz), 4.27 (t, 2H, J= 2.0 Hz), 4.27-4.22 (m, 4H), 4.16 (d, IH, J= 8.5 Hz), 3.78 (d, IH, J= 7.0 Hz), 3.71-3.67 (m, 4H), 3.65 (s, 4H), 3.36 (broad s, IH), 2.85 (dt, 2H, J= 7.0, 2.0 Hz), 2.81-2.67 (m, 2H), 2.68-2.63 (m, 4H), 2.52 (ddd, IH, J= 15.0, 10.0, 6.5 Hz), 2.48 (broad s, IH), 2.37 (s, 3H), 2.28 (m, 2H), 1.85 (m, IH), 1.84 (s, 3H), 1.73 (broad s, IH), 1.66 (s, 3H), 1.61 (s, 6H), 1.33 (s, 9H), 1.25 (s, 3H), 1.13 (s, 3H); ¹³C NMR (500 MHz, CDCl₃) δ 203.7, 174.4, 173.1 , 172.6, 172.2, 172.1, 171.6, 170.3, 167.1 , 155.5, 152.4, 142.6, 138.4, 136.3, 135.4, 133.8, 133.0, 130.3, 129.2, 129.0, 128.8, 128.2, 128.1, 126.8, 123.2, 1 15.0, 108.6, 84.5, 81.2, 81.1, 80.4, 79.1, 78.6, 76.5, 75.9, 75.0, 73.7, 72.5, 72.2, 70.6, 69.2, 64.0, 62.2, 58.6, 56,3, 52.3, 45.7, 43.2, 35.7, 35.6, 29.2, 29.1, 28.9, 28.8, 28.2, 26.8, 23.3, 22.7, 22.0, 14.9, 9.6; MS m/z 1487.5585 (M⁺+l).

Tetra(ethylene glycol) (1 mL, 5.8 mmol) and pyridine (0.47 mL, 5.8 mmol) were dissolved in DCM (4.6 mL) and the solution of TBSCl (0.873 g, 5.8 mmol) in DCM (2.3 mL) was added at O⁰C. The mixture was stirred at room temperature overnight. The reaction solution was concentrated and purified by flash chromatography (50% ethyl acetate in hexane) to give a colorless oil (717 mg, 40%). To the solution of this colorless oil (70 mg, 0.225 mmol), compound 3 (34 mg, 0.075 mmol) and DMAP (28 mg, 0.225 mmol) in DCM (4 mL) was added EDAC (44 mg, 0.225 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 20 mL), washed with saturated brine, and dried over NaSO₄. Then the mixture was vacuum filtered and rotary evaporated. Without purification, the residue was dissolved in the solution of HCl in ethanol (1%, 7.2 mL) at room temperature. After stirring for 15 minutes, the mixture was treated with saturated NaHCO₃ solution carefully and extracted with ethyl acetate (3X 20 mL). The combined organic phase was then washed with saturated brine, dried over NaSO₄, vacuum filtered, and rotary evaporated. The residue was purified by flash chromatography (5% methanol in DCM) to afford compound 11 (19.5 mg, 40.6% for two steps) as a white solid.

To the solution of docetaxel analog 8 (22 mg, 0.019 mmol), compound 11 (10 mg, 0.0156 mmol) and DMAP (6 mg, 0.047 mmol) in DCM (0.5 mL) was added EDAC (10 mg, 0.05 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. Then the mixture was vacuum filtered and rotary evaporated. After purified by flash chromatography (60% ethyl acetate in hexane), the product (16 mg, 0.009 mmol) was dissolved in pyridine (0.5 mL) and CH₃CN (0.25 mL). HF-pyridine (0.068 mL) was added to the solution at room temperature. The mixture was stirred at 62 ⁰C until the completion of the reaction (monitored by TLC). Then saturated NaHCO₃ solution was added to the reaction mixture dropwise. The mixture was extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. After vacuum filtration and rotary evaporation, the residue was then purified by flash chromatography (70% ethyl acetate in hexane) to give compound JQ206-105 (also referred to herein as "VT206-105") (8.4 mg, 35.2% for two steps) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 8.13 (d, IH, J = 1.5 Hz), 8.09 (d, 2H, J = 8.0 Hz), 7.98 (dd, IH, J= 8.5, 1.5 Hz), 7.91 (d, IH, J = 8.5 Hz), 7.60 (t, IH, J= 7.5 Hz), 7.49 (t, 2H, J = 7.5 Hz), 7.41-7.35 (m, 4H), 7.31 (tt, I H, J= 7.0, 1.5 Hz), 6.29 (s, IH), 6.22 (t, IH₁ J = 8.5 Hz), 5.65 (d, IH, J= 7.0 Hz), 5.37 (d, I H, J= 9.5 Hz), 5.25 (d, IH, J= 9.0 Hz), 4.93 (dd, IH, J= 9.0, 1.5 Hz), 4.68 (t, 2H, J= 2.0 Hz), 4.61 (m, IH), 4.38 (dd, IH, J=10.5, 7.0 Hz), 4.29 (d, IH, J= 8.0 Hz), 4.27 (t, 2H, J= 2.0 Hz), 4.27-4.21 (m, 4H), 4.15 (d, IH, J= 8.0 Hz), 3.78 (d, IH, J= 7.5 Hz), 3.71-3.67 (m, 4H), 3.65 (s, 4H), 3.65 (s, 4H), 2.85 (dt, 2H, J= 7.5, 1.5 Hz), 2.81-2.67 (m, 2H), 2.68-2.64 (m, 4H), 2.53 (ddd, IH, J= 15.0, 9.5, 7.0 Hz), 2.47 (broad s, IH), 2.37 (s, 3H), 2.28 (m, 2H), 1.86 (m, IH), 1.84 (s, 3H), 1.70 (broad s, IH), 1.65 (s, 3H), 1.61 (s, 6H), 1.32 (s, 9H), 1.24 (s, 3H), 1.13 (s, 3H); ¹³C NMR (500 MHz, CDCl₃) δ 203.6, 174.4, 173.1 , 172.6, 172.2, 172.1, 171.6, 170.3, 167.2, 155.5, 152.4, 142.6, 138.3, 136.3, 135.4, 133.8, 133.0, 130.3, 129.2, 129.0, 128.8, 128.2, 128.1, 126.8, 123.2, 123.1, 1 15.0, 108.6, 84.5, 81.1, 80.3, 79.1, 78.6, 76.5, 75.9, 75.0, 73.7, 72.5, 72.2, 70.7, 69.1, 64.0, 62.2, 58.6, 56.2, 52.3, 45.7, 43.3, 35.7, 35.5, 29.2, 29.1, 28.9, 28.8, 28.2, 26.8, 23.3, 22.7, 22.0, 14.9, 9.6; MS m/z 1531.5819 (M⁺H-I).

Penta( ethylene glycol) (2 mL, 9.5 mmol) and pyridine (0.767 mL, 9.5 mmol) were dissolved in DCM (7.5 mL) and the solution of TBSCl (1.43 g, 9.5 mmol) in DCM (3.8 mL) was added at 0° C. The mixture was stirred at room temperature overnight. The reaction solution was concentrated and purified by flash chromatography (70% ethyl acetate in hexane) to give a colorless oil (1.426 mg, 43%).

To the solution of this colorless oil (90 mg, 0.257 mmol), compound 3 (39 mg, 0.084 mmol) and DMAP (32 mg, 0.257 mmol) in DCM (4.6 mL) was added EDAC (50 mg, 0.257 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 20 mL), washed with saturated brine, and dried over NaSO₄. Then the mixture was vacuum filtered and rotary evaporated. Without purification, the residue was dissolved in the solution of HCl in ethanol (1%, 8.2 mL) at room temperature. After stirring for 15 minutes, the mixture was treated with saturated NaHCO₃ solution carefully and extracted with ethyl acetate (3X 25 mL). The combined organic phase was then washed with saturated brine, dried over NaSO₄, vacuum filtered, and rotary evaporated. The residue was purified by flash chromatography (5% methanol in DCM) to afford compound 12 (18.8 mg, 32.7% for two steps) as a white solid.

To the solution of docetaxel analog 8 (23 mg, 0.02 mmol), compound 12 (1 1 mg, 0.0161 mmol) and DMAP (6 mg, 0.05 mmol) in DCM (0.5 mL) was added EDAC (10 mg, 0.05 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. Then the mixture was vacuum filtered and rotary evaporated. After purified by flash chromatography (70% ethyl acetate in hexane), the product (17 mg, 0.0092 mmol) was dissolved in pyridine (0.52 mL) and CH₃CN (0.26 mL). HF-pyridine (0.071 mL) was added to the solution at room temperature. The mixture was stirred at 62 ⁰C until the completion of the reaction (monitored by TLC). Then saturated NaHCO₃ solution was added to the reaction mixture dropwise. The mixture was extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. After vacuum filtration and rotary evaporation, the residue was then purified by flash chromatography (70% ethyl acetate in hexane) to give compound JQ206-106 (also referred to herein as "VT206-106") (9.5 mg, 37.5% for two steps) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 8.13 (d, IH, J= 2.0 Hz), 8.09 (d, 2H, J= 8.0 Hz), 7.98 (dd, IH, J= 8.5, 2.0 Hz), 7.91 (d, IH, J= 8.5 Hz), 7.60 (tt, IH, J= 7.5, 1.5 Hz), 7.49 (t, 2H, J = 8.0 Hz), 7.42-7.35 (m, 4H), 7.31 (tt, IH, J= 7.5, 1.5 Hz), 6.29 (s, IH), 6.22 (t, IH, J = 8.0 Hz), 5.65 (d, IH, J= 6.5 Hz), 5.38 (d, IH, J= 8.5 Hz), 5.25 (d, I H, J= 8.0 Hz), 4.93 (dd, 1H, J= 9.5, 1.5 Hz), 4.68 (t, 2H, J= 2.0 Hz), 4.61 (m, lH), 4.38 (dd, IH, J=I 1.0, 7.0 Hz), 4.29 (d, IH, J= 8.5 Hz), 4.27 (t, 2H, J= 2.0 Hz), 4.26-4.21 (m, 4H), 4.15 (d, I H, J= 8.0 Hz), 3.78 (d, IH, J= 7.0 Hz), 3.71-3.67 (m, 4H), 3.65 (s, 4H), 3.65 (s, 4H), 3.64 (s, 4H), 2.84 (dt, 2H, J = 6.5, 1.5 Hz), 2.80-2.67 (m, 2H), 2.66-2.63 (m, 4H), 2.52 (ddd, I H, J= 14.5, 9.0, 6.0 Hz), 2.48 (broad s, IH), 2.37 (s, 3H), 2.28 (m, 2H), 1.85 (m, IH), 1.84 (s, 3H), 1.71 (broad s, IH), 1.65 (s, 3H), 1.61 (s, 6H), 1.32 (s, 9H), 1.24 (s, 3H), 1.13 (s, 3H); ¹³C NMR (500 MHz, CDCl₃) (5 203.6, 174.4, 173.1, 172.6, 172.2, 172.1, 171.6, 170.3, 167.1, 155.5, 152.4, 142.7, 138.4, 136.3, 135.4, 133.8, 132.9, 130.3, 129.2, 129.0, 128.8, 128.2, 128.1, 126.8, 123.2, 123.1 , 1 15.1, 108.7, 84.5, 81.1, 80.3, 79.1, 78.6, 76.5, 75.9. 75.0, 73.7, 72.4, 72.2, 70.7, 70.6, 69.1, 64.1, 64.0, 62.2, 60.5, 58.6, 56.2, 52.3, 45.7, 43.3, 35.7, 35.6, 29.2, 29.1, 28.9, 28.8, 28.2, 26.8, 23.3, 22.7, 22.0, 14.9, 9.6; MS m/z 1575.5980 (M⁺+l ).

To the solution of paclitaxel analog 5 (24 mg, 0.02 mmol), compound 10 (20 mg, 0.034 mmol) and DMAP (8 mg, 0.06 mmol) in DCM (0.6 mL) was added EDAC (12 mg, 0.06 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 10 mL), washed with saturated brine, and dried over NaSO₄. Then the mixture was vacuum filtered and rotary evaporated. After purified by flash chromatography (60% ethyl acetate in hexane), the product (14 mg, 0.0085 mmol) was dissolved in THF (0.6 mL) and HF-pyridine (0.14 mL) was added at 0 ⁰C. The mixture was stirred at room temperature until the completion of the reaction (monitored by TLC). Then saturated NaHCO₃ solution was added to the reaction mixture. The mixture was then extracted with ethyl acetate (3X 10 mL), washed with saturated brine, and dried over NaSO₄. After vacuum filtration and rotary evaporation, the residue was then purified by flash chromatography (70% ethyl acetate in hexane) to give compound JQ206-21 (also referred to herein as "VT206- 21 ") (7 mg, 22.8% for two steps) as a white solid: ¹H NMR (500 MHz, CDCI₃) δ 8.13 (d, I H, J = 2.0 Hz), 8.10 (dd, 2H, J= 8.0, 1.5Hz), 7.98 (dd, IH, J = 8.5, 2.0 Hz), 7.91 (d, I H₅ J= 8.5 Hz), 7.76 (dd, 2H, J = 8.0, 1.5 Hz), 7.61 (tt, IH, J = 7.5, 1.5 Hz), 7.52-7.46 (m, 5H), 7.43-7.38 (m, 4H), 7.34 (tt, IH, J= 7.5, 2.0 Hz), 7.08 (d, IH, J= 9.0 Hz), 6.17 (s, IH), 6.16 (dt, I H, J= 9.0, 1.5 Hz), 5.79 (dd, IH, J= 8.5, 2.5 Hz), 5.65 (d, IH, J= 7.0 Hz), 5.55 (dd, IH, J= 10.5, 7.5 Hz), 4.92 (dd, 1 H, J = 9.0, 1.0 Hz), 4.79 (d, 1 H, J = 2.5 Hz), 4.68 (d, 2H, J = 2.0 Hz), 4.30 (d, 1 H, J = 9.0 Hz), 4.27 (d, 2H, J= 2.0 Hz), 4.25-4.21 (m, 4H), 4.17 (d, IH, J= 9.0 Hz), 3.89 (d, IH, J = 7.0 Hz), 3.70-3.67 (m, 4H), 3.64 (s, 4H), 2.71-2.54 (m, 9H), 2.36 (s, 3H), 2.31 (m, 2H), 2.15 (s, 3H), 1.82 (m, IH), 1.81 (d, 3H, J= 1.0 Hz), 1.79 (s, 3H), 1.61 (d, 6H, J= 2.5 Hz), 1.19 (s, 3H), 1.15 (s, 3H); ¹³C NMR (500 MHz, CDCl₃) δ 201.9, 174.4, 172.6, 172.5, 172.2, 171.6, 170.4, 169.1, 167.1, 167.0, 152.4, 140.5, 138.1, 136.3, 135.4, 133.9, 133.8, 133.0, 132.0, 130.2, 129.1, 128.8, 128.4, 128.1, 127.2, 127.1, 123.2, 115.1, 108.6, 84.0, 81.1, 78.6, 76.5, 75.4, 74.3, 73.3, 72.2, 71.6, 70.6, 69.2, 69.1, 64.0, 63.8, 62.2, 56.2, 55.0, 52.3, 47.1, 43.3, 35.6, 33.4, 29.2, 28.9, 28.8, 26.6, 23.3, 22.6, 20.9, 14.7, 10.9; MS m/z 1555.4937 (M⁺+ Na)

13 To the solution of docetaxel analog 7 (87 mg, 0.082 mmol) in toluene (5 mL) was added glutaric anhydride (28 mg, 0.246 mmol) and DMAP (30 mg, 0.246 mmol). The mixture was stirred at 85-90⁰C for 5 days. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate and aqueous HCl solution (0.2%) was added carefully. The aqueous layer was then extracted with ethyl acetate (3X 20 mL). The combined organic phase was washed with saturated brine and dried over NaSO₄. After vacuum filtration and rotary evaporation, the residue was purified by flash chromatography (40% ethyl acetate in hexane) to afford white solid 13 (38 mg, 39%).

JQ206-85 To the solution of docetaxel analog 13 (38 mg, 0.032 mmol), compound 2 (34 mg, 0.096 mmol) and DMAP (12 mg, 0.096 mmol) in DCM (1.7 mL) was added EDAC (19 mg, 0.096 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. Then the mixture was vacuum filtered and rotary evaporated. Without purification, the residue was dissolved in pyridine (1.6 mL) and CH₃CN (0.8 mL). HF-pyridine (0.23 mL) was added to the solution at room temperature. The mixture was stirred at 62 ⁰C until the completion of the reaction (monitored by TLC). Then saturated NaHCO₃ solution was added to the reaction mixture dropwise. The mixture was extracted with ethyl acetate (3X 20 mL), washed with saturated brine, and dried over NaSO₄. After vacuum filtration and rotary evaporation, the residue was then purified by flash chromatography (50% ethyl acetate in hexane) to give compound JQ206-85 (also referred to herein as "VT206-85") (20.4 mg, 53.6% for two steps) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 8.13 (d, IH, J = 2.0 Hz), 8.10 (d, 2H, J = 7.5 Hz), 7.99 (dd, IH, J= 8.5, 2.0 Hz), 7.90 (d, IH, J= 8.5 Hz), 7.61 (tt, IH, J= 7.5, 1.5 Hz), 7.50 (t, 2H, J = 8.0 Hz), 7.42-7.35 (m, 4H), 7.32 (tt, IH, /= 7.0, 1.5 Hz), 6.30 (s, IH), 6.23 (t, IH, J= 3.5 Hz), 5.66 (d, IH, J= 7.5 Hz), 5.35 (d, IH, J= 9.5 Hz), 5.25 (d, IH, J= 8.0 Hz), 4.94 (dd, IH, J= 9.5, 2.0 Hz), 4.69 (t, 2H, J= 2.0 Hz), 4.62 (s, I H), 4.40 (m, I H), 4.30 (d, IH, J= 9.0 Hz), 4.29 (t, 2H, J= 2 Hz), 4.16 (d, I H, J= 9.0 Hz), 3.79 (d, IH, J= 7.0 Hz), 3.34 (d, IH, J= 3.5 Hz), 2.65-2.50 (m, 4H), 2.46 (d, I H, J =4.5 Hz), 2.37 (s, 3H), 2.27 (m, 2H), 2.04 (tt, 2H, J= 7, 7 Hz), 1.86 (m, IH), 1.84 (d, 3H, J= 1.0 Hz), 1.66 (s, 3H), 1.63 (s, 6H), 1.33 (s, 9H), 1.25 (s, 3H), 1.13 (s, 3H); ¹³C NMR (500 MHz, CDCl₃) δ 203.7, 174.4, 173.0, 172.2, 170.3, 167.1, 155.5, 152.4, 142.3, 138.2, 136.3, 135.4, 133.9, 133.8, 132.9, 130.3, 129.2, 129.0, 128.8, 128.2, 128.1, 126.8, 123.2, 123.1, 1 15.0, 108.6, 84.4, 81.1, 81.0, 79.1, 78.7, 76.5, 75.7, 75.0, 73.8, 72.4, 72.3, 62.2, 58.7, 56.3, 52.1, 45.7, 43.3, 35.7, 35.5, 33.1, 32.8, 29.2, 28.2, 26.8, 23.3, 22.7, 22.0, 20.0, 14.9, 9.7; MS m/z 1269.4652 (M⁺+l).

To the solution of docetaxel derivative 6 (105 mg, 0.094 mmol) in DCM (1 mL) was added TFA (0.48 mL) dropwise at 0⁰C. The mixture was stirred at O⁰C until the completion of the reaction (monitored by TLC). After careful addition of saturated NaHCO₃ solution, the mixture was extracted with ethyl acetate (3X 20 mL), washed with saturated brine, dried over NaSO₄, vacuum filtered and rotary evaporated. The residue was then purified by flash chromatography (40% ethyl acetate in hexane) to give a white solid product (65 mg, 68%).

This white solid (65 mg, 0.064 mmol) was dissolved in DCM (1 mL). Succinic anhydride (20 mg, 0.2 mmol) and DMAP (24 mg, 0.192 mmol) was added successively. The mixture was then stirred at room temperature overnight. After addition of water, the mixture was extracted with ethyl acetate (3X 20 mL), washed with saturated brine, dried over NaSO₄, vacuum filtered and rotary evaporated. The residue was then purified by flash chromatography (40% ethyl acetate in hexane) to give a product (31 mg, 43.8%) as white solid.

The resulting product (31 mg, 0.028 mmol) was dissolved in ethanol (2.6 mL) and hydrazine monohydrate (0.29 mL) was added. The mixture was stirred at room temperature overnight. After the addition of saturated NH₄Cl solution, the mixture was extracted with ethyl acetate (3X 25 mL), washed with saturated brine, and dried over NaSO₄, followed by vacuum filtration and rotary evaporation. The residue was then purified by flash chromatography (50% ethyl acetate in hexane) to afford 14 (12 mg, 40%) as white solid.

To the solution of compound 14 (12 mg, 0.011 mmol), compound 2 (12 mg, 0.034 mmol) and DMAP (4.2 mg, 0.034 mmol) in DCM (0.6 mL) was added EDAC (6.6 mg, 0.034 mmol). The mixture was stirred at room temperature overnight. After the addition of water, the mixture was extracted with ethyl acetate (3X 15 mL), washed with saturated brine, and dried over NaSO₄. Then the mixture was vacuum filtered and rotary evaporated. After purified by flash chromatography (50% ethyl acetate in hexane), the product (15 mg, 0.01 1 mmol) was dissolved in pyridine (0.6 mL) and CH₃CN (0.3 mL). HF-pyridine (0.081 mL) was added to the solution at room temperature. The mixture was stirred at 62 ⁰C until the completion of the reaction (monitored by TLC). Then saturated NaHCO₃ solution was added to the reaction mixture dropwise. The mixture was extracted with ethyl acetate (3X 20 mL), washed with saturated brine, and dried over NaSO₄. After vacuum filtration and rotary evaporation, the residue was then purified by flash chromatography (100% ethyl acetate) to give compound JQ206-92 (also referred to herein as "VT206-92") (8.7 mg, 68.5% for two steps) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 8.1 1 (d, IH₁ J= 2.5 Hz), 8.09 (dd, 2H, J= 8.5, 1.0 Hz), 7.96 (dd, I H, J = 8.5, 2.5 Hz), 7.89 (d, IH, J= 8.5 Hz), 7.60 (tt, IH, J= 7.5, 1.5 Hz), 7.50 (t, 2H, J = 7.5 Hz), 7.41- 7.33 (m, 4H), 7.32 (tt, IH, J = 6.5, 2.0 Hz), 6.52 (d, IH₁ J= 9.0 Hz), 6.22 (dt, IH, J= 9.0, 1.5 Hz), 5.67 (d, IH, J= 7.0 Hz), 5.56 (dd, IH, J= 9.0, 2.5 Hz), 5.19 (s, IH), 4.91 (dd, IH, J= 9.5, 2.0 Hz), 4.65 (d, IH, J = 2.0 Hz), 4.53 (dt, IH, J= 16.0, 2.0 Hz), 4.32 (dt, I H₁ J= 16.0, 2.0 Hz), 4.27 (d, IH, J= 9.0 Hz), 4.23-4.20 (m, 3H), 4.20 (d, IH, J= 8.5 Hz), 3.88 (d, I H, J= 6.5 Hz), 3.49 (broad s, IH), 2.71-2.64 (m, IH), 2.59-2.42 (m, 4H), 2.34 (s, 3H), 2.26 (dd, 2H, J= 9.0, 6.5 Hz), 2.1 1 (broad s, IH), 1.83 (m, IH), 1.83 (d, 3H, J= 0.5 Hz), 1.74 (s, 3H), 1.57 (s, 6H), 1.24 (s, 3H), 1.12 (s, 3H); ¹³C NMR (500 MHz, CDCIj) 5 21 1.4, 174.3, 172.8, 172.4, 170.8, 170.3, 166.9, 152.4, 138.3, 138.0, 136.3, 136.2, 135.4, 133.8, 130.3, 129.4, 129.0, 128.8, 128.3, 128.1 , 126.9, 123.2, 123.1, 1 15.0, 108.6, 84.2, 81.2, 81.1, 78.7, 78.3, 76.7, 74.9, 74.6, 73.2, 72.8, 72.1, 62.2, 57.7, 54.6, 52.4, 46.5, 43.2, 37.0, 36.0, 30.4, 29.1, 29.0, 26.6, 23.2, 22.7, 20.9, 14.4, 10.0; MS m/z 1 155.5883 (M⁺+ 1).

Results

All the compounds of Example 2 were tested for their cytotoxicity against the A2780 ovarian cancer cell line; the results of these assays are given in Table 3. Compounds VTl 92- 219206-105, and 206-106 were the most cytotoxic, with cytotoxicities three to sevenfold greater than that of paclitaxel. Compounds VT192-297 and VT192-300 were about as cytotoxic as paclitaxel, and all the other AR-linked compounds were significantly less cytotoxic than paclitaxel.

Table 3. A2780 Bioassay Data for Synthetic Taxane-AR ligands

In addition to cytotoxicity determinations, the analysis of the first four compounds included tubulin polymerization activity, AR binding activity and analysis to test for concomitant binding. Taxanes bind tubulin polymers, but not tubulin monomers. Therefore, analysis for tubulin polymerization serves as a surrogate for tubulin binding. Analysis of compounds VT192-183, -204, -210 and -219 for tubulin polymerization by turbidimetry showed that compound 219 has the best activity and is comparable to paclitaxel (FIGs. 10a- 10c).

AR binding affinities were assessed using a tritiated mibolerone binding assay. The Ki of compounds VT-192-183, -204, -210 and -219 are 13798 (+/- 1317), 5820 (+/- 1952), 12096 (+/- 2481) and 15660 (+/- 103), respectively. DHT was used as a control (Ki = 3.4 +/- 3.4). The affinities of these compounds for AR are therefore relatively low.

The compounds also were tested on 293 cells expressing a (GFP-AR) construct by confocal microscopy. No significant decrease in nuclear GFP-AR by any of these compounds was observed.

Gene expression analysis of the compounds that have the best tubulin binding and tubulin polymerization activity - the compounds having the linkage on the 10-position of the taxane - was also conducted (see FIG. 1 1). The gene expression signatures of paclitaxel and compound 85 clustered together and both had similar levels of survivin induction. Survivin is a prosurvival gene that is induced with therapy with paclitaxel, is a poor prognostic feature when expressed highly in tumors and is a mediator of resistance to chemotherapy. The lack of survivin induction with these novel taxane analogs is therefore an important finding for taxane therapy that does not induce an important mechanism of drug resistance for patients with prostate, breast and lung cancers.

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In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention.

Claims

We claim:

1. A compound, or a pharmaceutically acceptable salt thereof, comprising a structure represented by formula II:

X - alkynylene linker - Z

wherein X is a steroid hormone receptor binding moiety; and

Z is (i) an extranuclear element binding moiety or (ii) a blocking moiety that disrupts the interaction between a steroid hormone receptor and a steroid hormone receptor coactivator.

2. A compound, or a pharmaceutically acceptable salt thereof, comprising a structure represented by formula I:

X - L - Z

wherein X is a steroid hormone receptor binding moiety;

Z is (i) a moiety that binds to an extranuclear element or (ii) a blocking moiety that disrupts the interaction between a steroid hormone receptor and a steroid hormone receptor coactivator; and

L is a linking group covalently bound to X and Z, wherein the linking group is physiologically-noncleavable and has a structural length and rigidity sufficient to allow the compound to bind to the steroid hormone receptor and (i) the extranuclear element or (ii) the blocking moiety.

3. The compound of claims 1 or 2, wherein X is a Type I steroid hormone receptor binding moiety.

4. The compound of any one of claims 1 to 3, wherein Z is a colchicinoid.

5. The compound of claim 2 wherein L includes at least one carbon-carbon triple bond.

6. The compound of any one of claims 1 , 2, 4 or 5, wherein X is selected from an androgen receptor antagonist, an estrogen receptor antagonist, a progesterone receptor antagonist, a selective androgen receptor modulator, a selective estrogen receptor modulator, an androgen receptor agonist, an estrogen receptor agonist, or a progesterone receptor agonist.

7. The compound of claim 6, wherein X is an androgen receptor antagonist.

8. The compound of claim 6, wherein X is a selective estrogen receptor modulator.

9. The compound of any one of claims 1, 2, 4 or 5, wherein X is selected from cyanonilutamide, nilutamide, fiutamide, hydroxyflutamide, bicalutamide, megestrol acetate, bazedoxifene, clomifene, fulvestrant, raloxifene, tamoxifen, toremifene, mifepristone, andarine, ostarine, prostarin, andromustine, BMS-564929, spironolactone, eperenone, ketoconazole, or an analog thereof.

10. The compound of claim 1 or 2, wherein Z is a colchicinoid and X is selected from cyanonilutamide, nilutamide, fiutamide, hydroxyflutamide, bicalutamide, megestrol acetate, or an analog thereof.

1 1. The compound of claim 2, wherein L has a structure that does not undergo cleavage under intracellular or extracellular conditions.

12. The compound of claim 1, wherein the extranuclear element is selected from tubulin, actin, FK binding proteins, cyclophilin, cellular lipid or cellular sugar.

13. The compound of any one of claims 1-12, wherein Z is a tubulin binding moiety.

14. The compound of any one of claims 1 -12, wherein Z is selected from a colchicinoid, a taxane or an epothilone.

15. The compound of any one of claims 1, 3, 4, 6-10 and 12-14, wherein the compound has the structure: X-(C(R')₂)_n-C≡C-(C(R²)₂)_m-Z

wherein R¹ and R² are each independently H, alkyl, aryl, hydroxyl, alkoxy, carboxyl, alkoxycarbonyl or halogen; and n and m are each independently an integer from 0 to 5.

16. A compound, or a pharmaceutically acceptable salt thereof, comprising a structure represented by formula III:

W - alkynylene linker - Y

wherein W is selected from cyanonilutamide, nilutamide, flutamide, hydroxyflutamide, bicalutamide, megestrol acetate, bazedoxifene, clomifene, fulvestrant, raloxifene, tamoxifen, toremifene, mifepristone, andarine, ostarine, prostarin, andromustine, BMS-564929, spironolactone, eperenone, ketoconazole, or an analog thereof; and

Y is a colchicinoid or a taxane.

17. The compound of any one of claims 1-14 or 16, wherein L or the alkynylene linker is - C≡C-.

18. The compound of any one of claims 1-14 or 16, wherein L or the alkynylene linker includes -C≡C-.

19. The compound of any one of claims 1-14 or 16, wherein L or the alkynylene linker is selected from:

-CH₂-O-C≡C-;

-C(O)O-C≡C-; or

R¹⁰-C(O)O-C≡C- , wherein R¹⁰ is -C(O)-(CH₂),- or

R¹⁰ is -C(O)-(CH₂)₂-C(O)-(O-(CH₂)₂)_y-O-C(O)-(CH₂)₂)-, wherein x is 1 to 20, 1 to 10, or 2 to 5 and y is 1 to 100, 2 to 100, 2 to 50, or 2 to 20.

20. The compound of any one of claims 1-14 or 16, wherein L or the alkynylene linker is selected from ethynylene, 1 -propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 3- butynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, 4-pentynylene, 1-hexynylene, 2- hexynylene, 3-hexynylene, 4-hexynylene, 2,4-hexadiynylene, 5-hexynylene, 1-heptynylene, 2- heptynylene, 3-heptynylene, 4-heptynylene, 5-heptynylene, 6-heptynylene, 1-octynylene, 2- octynylene, 3-octynylene, 4-octynylene, 5-octynylene, 6-octynylene, or 7-octynylene.

21. The compound of any one of claims 1-14 or 16, wherein L or the alkynylene linker does not include a salicylamide N-Mannich base structure.

22. The compound of any one of claims 1-14 or 16, wherein L or the alkynylene linker is not attached to either the steroid hormone receptor binding moiety or the tubulin binding moiety via a salicylamide N-Mannich base structure.

23. The compound of any of claims 16 to 22, wherein W is cyanonilutamide.

24. The compound of claim 23, wherein the alkynylene linker is R¹⁰-C(O)O-C≡C- , wherein

or R¹⁰ is -C(O)-(CH2)2-C(O)-(O-(CH2)2)y-O-C(O)-(CH₂)2)-, wherein x is 1 to 20, 1 to 10, or 2 to 5 and y is 1 to 100, 2 to 100, 2 to 50, or 2 to 20.

25. The compound of any one of claims 16 to 24, wherein Y is colchicine.

26. The compound of any one of claims 16 to 24, wherein Y is paclitaxel.

27. The compound of any one of claims 16 to 24, wherein Y is docetaxel.

28. The compound of claim 16, wherein the compound is selected from:

wherein n is O to 20; X is a halogen, OCH₃ or N₃; and R is an aryl group, a heterocyclic group, or OR' wherein R' is C_rCi₂ straight-chain or branched alkyl.

29. The compound of any of claims 1-28, wherein L or the alkynylene linker also includes a polyethylene glycol moiety.

30. A pharmaceutical composition comprising a therapeutically effective amount of any one of the compounds of claims 1 to 29 and a pharmaceutically acceptable carrier.

31. A method for inhibiting in a subject a disease associated with and dependent upon hormone receptor activity or lack of activity, comprising administering to a subject a therapeutically effective amount of any one of the compounds of claims 1 to 29.

32. The method of claim 31, wherein the disease is hormone-resistant cancer.

33. The method of claim 32, wherein the cancer is breast cancer.

34. The method of claim 32, wherein the cancer is prostate cancer.

35. The method of claim 32, wherein the cancer is endometrial cancer.

36. The method of claim 31, wherein the disease is androgenetic alopecia, acne, benign prostatic hyperplasia, osteoporosis, cardiovascular disease, muscle wasting, anemia, sexual dysfunction, endometriosis, polycystic ovarian syndrome, leukemia, lymphoma, multiple myeloma, or hypertension.

37. The method of any one of claims 33, wherein X is a steroidal estrogen, a selective estrogen receptor modulator, or an estrogen receptor antagonist.

38. The method of claim 34, wherein X is an androgen receptor antagonist, an androgen agonist, or a selective androgen receptor modulator.

39. A method for inhibiting in a subject a neoplasm that has developed a resistance to chemotherapeutic treatment, comprising administering to a subject a therapeutically effective amount of any one of the compounds of claims 1 to 29.

40. A method for anchoring a steroid hormone receptor in cell cytoplasm, comprising: contacting the cell with any one of the compounds of claims 1 to 29, thereby binding the compound to an extranuclear element in the cell cytoplasm.

41. A method for binding a steroid hormone receptor binding agent to tubulin, comprising: contacting any one of the compounds of claims 1 to 29 with tubulin, thereby binding the compound of claim 1 to the tubulin.