WO2014008159A1 - Selective estrogen receptor degraders for treatment of tamoxifen resistant tumors - Google Patents

Abstract

Compositions and methods for covalently bonding a polycarboxylic fatty acid and a SERD (selective estrogen receptor degrader) to form fatty acid-drug conjugate compounds are described. The conjugate compounds are useful for increasing solubility of the SERD as well as targeting the SERD to solid tumors. In particular, the compounds are surprisingly efficacious against tamoxifen-resistant tumors. These types of tumors are recalcitrant to existing therapies and often signal the end stage of cancer.

Description

SELECTIVE ESTROGEN RECEPTOR DEGRADERS FOR

TREATMENT OF TAMOXIFEN RESISTANT TUMORS

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under NIH R01 CA 126825 awarded by the National Institutes of Health and NCBC# 201 l-CFG-8003. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure is directed to fatty acid derivatives and analogs of selective estrogen receptor degraders (SERDs), in particular those that contain malonic, succinic, and glutaric acid moieties having a pendant C_8-25 alkyl chain.

BACKGROUND OF THE INVENTION

The estrogen receptor ("ER") is a ligand-activated transcriptional regulatory protein that mediates induction of a variety of biological effects through its interaction with endogenous estrogens. Endogenous estrogens include 17 -estradiol and estrones. ER has been found to have two isoforms, ER-a and ER-β. Estrogens and estrogen receptors are implicated in a number of diseases or conditions, such as breast cancer, ovarian cancer, colon cancer, prostate cancer, endometrial cancer, uterine cancer, as well as others diseases or conditions.

The gold standard treatment in ER+ breast cancer is oral tamoxifen, a selective estrogen receptor modulator (SERM). Over time or by chance mutation, ER+ breast cancer not only becomes resistant to tamoxifen as tomoxifen becomes an agonist which induces proliferation. Unfortunately, there is only one approved hormonal medication for tamoxifen resistant cancer, the selective estrogen receptor degrader (SERD) fulvestrant. Due to solubility issues, it has to be administered as an IM injection in oil which is extremely painful and unpleasant for the patients and its intrinsic solubility precludes it from reaching efficacious serum concentrations.

What is needed, therefore, is an alternative treatment for patients suffering from tamoxifen-resistant cancers. SUMMARY OF THE INVENTION

Compositions and methods for covalently bonding a polycarboxylic fatty acid and a SERD (selective estrogen receptor degrader) to form fatty acid-drug conjugate compounds are described. The conjugate compounds are useful for increasing solubility of the SERD as well as targeting the SERD to solid tumors. In particular, the compounds have been shown to be surprisingly efficacious against tamoxifen-resistant tumors. These types of tumors are recalcitrant to existing therapies and often signal the end-stage of cancer. Currently, there is only one approved drug for use in patients suffering from tamoxifen-resistant cancer. Unfortunately, this drug, fulvestrant, is not easily administered and its efficacy is lacking. Accordingly, the compounds and methods disclosed herein address the need for alternative and improved drugs for treating tamoxifen-resistant tumors.

In an embodiment, the present disclosure is directed to long-chain fatty acid derivatives of SERDs. When conjugated in this way to a SERD, the fatty acid molecule contains one free carboxylic acid, ester or other inorganic acid anion(s). The anionic moiety of the SERD is believed to be responsible for degradation the estrogen receptor. This is because the ligand binding domain of the estrogen receptor has an aspartic acid at residue 351. The repulsion caused by the two anions leads to hindered binding of co-activators.

In contrast, tamoxifen contains a cationic moiety which favorably interacts with the aspartic acid at residue 351. This interaction has been shown to increase levels of transforming growth factor-a which explains the estrogenic like activity tamoxifen can exhibit. Tamoxifen binds ER triggering a structural modification that makes it more likely to attract co-repressors and less likely to bind co-activators in a reversible manner. Unlike the SERDs, the SERM tamoxifen can act as an estrogen receptor agonist.

SERDs on the other hand cause internalization of the receptor and degradation via the ubiquitin pathway which is an irreversible step. The SERD compounds described herein are receptor degraders and are effective at slowing growth of or regressing tamoxifen-resistant tumors which can lead to remission or progression free survival with fewer side effects compared to other antiestrogen therapies. Additionally, the long-chain fatty acid- SERD conjugate compounds can have improved biopharmaceutical properties and a high therapeutic index over the parent SERD compound. In this embodiment, the subject matter disclosed herein is directed to long- chain fatty acid-SERD conjugate compounds.

Another embodiment is directed to treating a tamoxifen-resistant tumor in a mammal in need thereof by administering a long-chain fatty acid-SERD conjugate compound as described herein.

Another embodiment is directed to the use of long-chain fatty acid molecules to prepare fatty acid-drug SERD conjugate compounds.

Another embodiment is directed to the use of the long-chain fatty acid to increase solubility, half-life and/or efficacy of the SERD.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 A & B depict the effect of Antiestrogens on MCF7 xenograft tumor growth. (A) Exponential growth curve fits indicating mean tumor volume ± SEM for all remaining tumors at the indicated time-point. (B) Kaplan-Meir analysis indicating the time for individual tumors to reach a size of 0.7cm³. Dashes indicate mice that were either withdrawn from the study or those whose tumors failed to reach a size of 0.7cm³.

Figure 2A & B depict the effect of Antiestrogens on tamoxifen-resistant MCF7 (TAMR) xenograft tumor growth. (A) Exponential growth curve fits indicating mean tumor volume ± SEM for all remaining tumors at the indicated time-point. (B) Kaplan- Meir analysis indicating the time for individual tumors to reach a size of 0.5cm³.

Dashes indicate mice that were either withdrawn from the study or those whose tumors failed to reach a size of 0.5cm³.

Figures 3-15 depict individual plots compared to E2-control.

Figures 16-18 depict correlation between uterine weight and tumor doubling time.

Figures 19-21 depict NMR spectra for several conjugate compounds as described herein.

DETAILED DESCRIPTION

New drug compounds, formulations and methods of their use against tamoxifen-resistant tumors are provided herein. It has been surprisingly found that the conjugate compounds described herein are effective in slowing tumor growth in tamoxifen-resistant tumors. Estrogen receptors (ER) serve not only as a diagnostic marker indicator of appropriate treatment for breast cancer, but also as a therapeutic target to deter tumor growth. While the majority of ER-positive tumors initially respond to endocrine based therapies targeting the ER signaling axis at the level of ligand availability (aromatase inhibitors) or the receptor itself (tamoxifen), it is almost inevitable that resistance to tamoxifen and aromatase inhibitors will develop.

Despite the development of resistance, ER signaling remains intact, and continues to represent a viable drug target. However, available ER targeted therapies appropriate for use in the relapsed, advanced, or metastatic setting are currently limited to SERDs, such as fulvestrant (FASLODEX^®), a drug with relatively poor efficacy, limited in part by its pharmacokinetic properties. Fulvestrant is the only approved SERD against hormone receptor-positive metastatic breast cancer in postmenopausal women with disease progression following anti-estrogen. Therefore, the development of an alternative therapy with improved bioavailability, solubility and/or

pharmacokinetic profile is of particular interest and would serve an unmet need.

In one embodiment, the subject matter disclosed herein is directed to a compound comprising a long-chain fatty acid covalently bound to a SERD drug having the followi

and

wherein, A is a residue of a SERD, p is from one to 500, provided that if the linker L is other than ethylene glycol then p is zero or one, L is a linker, R¹ is a C₈-₂₅ alkyl, R² is a hydroxyl or Ci_-4 alkoxy, and x and y are each independently zero, one or two. Most preferably, both x and y are one, which is shown in Formula la.

Formulae I and la depict a carbon-carbon bond "™" that may be a single bond or double bond. As an example, when a long-chain alkyl derivative of maleic anhydride is used in the synthesis ound,

wherein R is as described in Formula I, one of "— " in the conjugate compound will be a double bond.

In preferred embodiments, however, each "— " is a single bond as shown in Formulae Γ and la' as follows:

Unlike selective estrogen receptor modulators (SERMs) like tamoxifen, selective estrogen receptor degraders (SERDs) are not ER agonists which limits side effects throughout the body such as endometrial hyperplasia or uterotrophy, bone demineralization, thromboembolic events and depleted serum lipids which makes them much more tolerable therapies. Useful SERDs include any known compounds that are capable of degrading the estrogen receptor and that have a nucleophilic group or can be modified to contain a nucleophilic group that is amenable to conjugation with a long- chain fatty acid as described herein. These compounds include those described in PCT/US201 1/039669, published as WO 201 1/156518, and U.S. Patent Appl. Pub. No. 2012/0071535, both of which are incorporated herein by reference in their entireties. Those of skill in the art are well aware of the mechanism by which SERDs can degrade an estrogen receptor.

Preferably, the SERD is selected from the group consisting NL3m and NL4. These compounds have the following structures:

These SERDs can isomerize around the central olefin to form either the (E) or (Z) isomer. While both isomers retain activity, one isomer tends to have a higher affinity and consequently a greater activity in degrading the estrogen receptor. For NL3, the (Z) isomer is preferred, whereas for NL4 the (£) isomer is preferred. The presence of the phenol in NL4 adds a degree of instability and allows E/Z isomerization in an equilibrative manner. This isomerization is believed to be photocatalyzed. The E/Z isomers of both NL4 and NL4-PDG have been separated by a long gradient on an analytical CI 8 reversed phase HPLC column. When dissolved in DMSO and exposed to a benchtop UV lamp, isometric equilibrium was achieved within days. To prevent this isomerization after purification, it is recommended the compounds be stored as a dry powder under an inert atmosphere in an amber vial or vial coated with aluminum foil.

(Z) NL3 (E) NL3

Most preferably, the SERD is NL4.

In particular, the compound NL3m is an example of a SERD that has been modified to contain an appropriate nucleophilic group. Schemes 3-5 depict a modification to a SERD to insert an appropriate chemical handle for conjugation to a long-chain fatty acid. Using the methods disclosed herein, those of skill in this field would be able to modify appropriate SERDs to facilitate the conjugation of the modified SERD with a long-chain fatty acid as described herein.

A is a residue of a SERD. The term "residue" refers to the SERD that has been covalently bonded to the long chain fatty acid directly or through a linker.

Accordingly, useful compounds are those where A is a residue of a SERD that has been covalently bonded to a linker or the long chain fatty acid.

Useful compounds include those where R¹ is a C_8-25 alkyl. Preferably, R¹ is a Cio_-20 alkyl. More preferably, R² is a C12-16 alkyl. Most preferably, R¹ is a C_{] 5} alkyl.

Useful compounds include those where R² is a hydroxyl or Ci _-4 alkoxy. When R is a Ci_-4 alkoxyl, it can be methoxy, ethoxy, rc-propoxy, -propoxy, «-butoxy, sec- butoxy or t-butoxy. When R² is a Ci_-4 alkoxyl, it is preferably methoxy or ethoxy. Most preferably, R is hydroxyl. Useful compounds include those where p is one or zero. In compounds where p is zero, the long chain fatty acid is bound directly to the SERD residue. In some embodiments, compounds where p is one result in the long chain fatty acid being covalently bound to a linker, L. The linker, L, is covalently bound to the SERD (A) through an available chemical handle on the SERD. In a preferred attachment, the linker is bound to the SERD as shown below:

SERD residue Long chain

fatty acid moiety

The linker, L, can be any known linker useful for covalently binding two separate moieties of a conjugate. Preferably, L is selected from the group consisting of

-}-0 0-|-

\— / , (ethylene glycol)

i

In compounds where L is i, p can be an integer from one to 500. In these compounds, a useful value of p is from 1 to 250. Preferably, p is from 1 to 100. Also preferred in these compounds are those compounds where p is from 1 to 50. More preferably, p is from 1 to 25. Most preferably, in these compounds, p is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1,

12, 13 ,14 or 15.

Useful compounds include compounds having the following formulae and structures:

wherein, Rl and R2 are as described above in the useful and all preferred embodiments;

a most preferred compound is (E) NL4-PDG;

and

wherein, L, Rl and R2 are as described above in the useful and all preferred

embodiments and R is hydroxyl;

(NL3eg-PDG)

(NL3m-PDG) The long chain fatty acid has at least one free carboxylic acid or carboxylate group. Preferably, the fatty acid is a dicarboxylic acid. More preferably, the fatty acid is derived from an anhydride. As used herein, the term "fatty acid" refers to C]_0-25 alkyl fatty acids and derivatives, as well as anhydrides of dicarboxylic acids. Preferably, the alkyl chain has from 12 to 20 carbons. More preferably, the alkyl chain has from 14 to 16 carbons. Most, preferably, the chain has 15 carbons. Useful fatty acids include any alkyldioic acid, which is a straight alkyl or alkenyl chain with carboxylates at the distal ends which include; malonic acid, succinic acid, maleic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid and the like. When a

polycarboxylic acid with a long alky chain is used in derivatizing the drug molecule, the resulting conjugate will carry a free -COOH group. Dicarboxylic acids such as malonic, succinic, and glutaric acids are useful and their simple derivatives that contain one long alkyl chain in which the number of carbons varies from 8 to 25, preferably 8 to 20, 10 to 20, 12 to 20 or 14 to 16.

When the conjugate compound contains, for example, a malonic acid moiety, in Formula I, x and y will both be zero. When the compound conjugate contains, for example, a succinic acid moiety, in Formula I, one of x and y will be zero and the other will be one. When the compound conjugate contains, for example, a glutaric acid moiety, in Formula I, x and y will both be one. Accordingly, those of skill in the art will readily be able to determine the value of x and y depending on which alkyldioic acid is employed. Any known alkyldioic acid can be used.

In addition to these dicarboxylic acid derivatives, any compounds that contain three or more -COOH groups can be used for the same purpose. Examples may include; citric acid, tricarboxylic acid and its derivatives such as beta- methyltricarboxylic acid, and 1,2,3,4-butanetetracarboxylic acid. Cyclic dicarboxylic acids such as camphoric acid and cyclic 1,3,5-cyclohexanetricarboxylic acid can also serve the same purpose. Mixed di- or multi-acids containing an inorganic acid are also included; a naturally occurring example is phosphorylatedN-acetyltyrosine, while sulfate esters of a hydroxy-containing carboxy acid is an example of synthetic in origin. Most preferably, the fatty acid is glutaric acid, in particular 3-pentadecylglutaric anhydride (PDG). The preferred fatty acid moiety is provided by the compound having the structure

wherein R' is a substituted or unsubstituted Ci_0-25 alkyl or Ci_0-25 alkylenyl. Preferably, R' is Ci_2-2o alkyl. More preferably, R' is Ci_4-i₆ alkyl. Most preferably, the compound is 3-pentadecylglutaric anhydride as discussed in PCT/US201 1/020221, published as WO 201 1/085000, incorporated herein by reference in its entirety. The terms "alkenyl" and "alkylenyl", when used alone or in combination, embraces linear or branched aliphatic chains having at least one carbon-carbon double bond between two adjacent carbon atoms. Examples include, without limitation, ethenyl, propenyl, butenyl and 4- methylbutenyl.

The present formulations and processes utilize the conjugation chemistry described herein. Importantly, the -COOH function of the fatty acid remains intact in the final conjugate. Preferably, the fatty acid molecule has more than one carboxylic acid moiety. When the fatty acid molecule contains more than one carboxylic acid moiety, for preparing the conjugate compounds the preferred form of the fatty acid molecule is the anhydride. Preparation of anhydrides from dicarboxylic acids is well known in the art.

In another embodiment, the subject matter disclosed herein is directed to a method of treating a mammal suffering from a tamoxifen-resistant cancer. In an aspect of this embodiment, an effective amount of a compound of Formuale I- VI is administered to a mammal in need thereof. Since there is currently only one approved drug for such cancers, this embodiment is particularly useful and needed as therapy to treat such cancers. As used herein, a mammal "in need thereof refers to a patient that has been diagnosed as having tamoxifen-resistant tumor cells. Alternatively, it may be determined that the patient is susceptible to a cancer that is likely to become tamoxifen- resistant. In particular, the conjugate compounds can be useful in treating metastatic breast cancer that is tamoxifen-resistant in post-menopausal women.

In another embodiment, the subject matter disclosed herein is directed to pharmaceutical formulations comprising a compound of Formulae I- VI as disclosed herein. The pharmaceutical formulations comprise a compound of Formulae I- VI and at least one pharmaceutically acceptable excipient and/or diluent.

In another embodiment, the subject matter disclosed herein is directed to a method of increasing the half-life, solubility and/or efficacy of a SERD. In this embodiment, a SERD is covalently bound to a long chain fatty acid as disclosed herein thereby forming a compound having an increased desirable property. In the methods directed to solubilizing a SERD, the methods comprise contacting the long-chain fatty acid-SERD conjugate conjugate described herein with a medium in which said conjugate is to be solubilized. The medium can be any liquid. Preferably, the medium is serum. In a preferred embodiment, the conjugate has a 100-fold increase in solubility compared to the solubility of the drug alone in a particular medium. More preferably, the conjugate has a 200-fold increase in solubility. Most preferably, the conjugate has a 250-fold increase in solubility.

In an embodiment, the present disclosure is directed to a method of preparing the long-chain fatty acid conjugate of Formulae I-VI and any intermediates. The method of preparing a compound of Formulae I-VI comprises contacting the SERD with a long-chain fatty acid or anhydride thereof wherein the desired conjugate compound is prepared. In an aspect of this embodiment, a method of conjugation is via a lipid which allows a facile single-pot synthesis of conjugate from drug. In particular, 3-pentadecylglutaric anhydride (PDG) chemistry allows for any drug containing a nucleophile to potentially be conjugated as described herein.

Any SERD can be used so long as it has a nucleophilic group or can be modified to contain a nucleophilic group. By nucleophile it is meant an alcohol, a thiol, a primary amine or secondary amine. These nucleophiles can be synthesized on the SERD if necessary. These modifications can be reversible thus generating prodrugs which will readily react with PDG that can be subsequently cleaved back to the intact drug. As an example, carboxylic acids are non-nucleophilic but common to many drugs. The formation of an ester using ethanolamine would yield a prodrug through a readily cleavable ester, as well as imparting a free amine which could react with PDG. The following schemes depict synthetic routes for preparing fatty acid molecules or conjugates as described herein. Scheme 1 depicts a synthetic route for preparing PDG (3-pentadecylglutaric anhydride).

Scheme 1

Scheme 2 depicts a synthetic route for preparing NL4-PDG.

Scheme 2

Scheme 3 depicts a one-step synthesis of NL3eg-PDG.

Scheme 3 Scheme 4 depicts a synthetic route for preparing NL3paba-PDG.

Scheme 4

Scheme 5

The long-chain fatty acid-SERD conjugate compounds and formulations containing them can utilize in vivo properties and functions of serum albumin. First, albumin circulates for a long time with ti_/2 of 19 days in humans. Secondly, it carries tightly bound fatty acids. Thirdly, when the drug formulation is a cancer therapy and when the tumor is growing, the protein accumulates and degrades mainly in the tumor.

Albumin of MW 66 kDa is the most abundant protein in the serum, providing osmotic pressure to blood vessel against hydrostatic pressure from the heart. It also serves as a natural carrier for a variety of xenobiotics as well as water-insoluble endogenous substances such as fatty acids. Its serum concentration is close to 50 mg/ml (5% or 0.75 mM) while it is -16 mg/ml (1.6 % or 0.24 mM) in the interstitial tissues. Since albumin provides six rather specific binding sites for fatty acid, the effective concentration in terms of fatty acid-binding site is much higher. In short, the protein acts as a sponge for hydrophobic xenobiotics and 99.9% of all fatty acids in the serum are in the state of bound to albumin. While structurally and functionally quite different from immunoglobulin IgG of MW 150 kDa, there are many similarities between the two major proteins in kinetics of synthesis and catabolism. IgG is second only to albumin in serum abundance at ~ 12 mg/ml. The size of these proteins makes them candidates for the so-called enhanced permeability and retention (EPR) effect. Briefly, these molecules move mainly by solvent drag (i.e., convection) through the bloodstream. When approaching a tumor, the pressure gradient pushes these macromolecules through a leaky vasculature into the tumor periphery. The tumor core is of higher fluid pressure preventing any convective penetration deeper into the tumor. With no ability to move back to the circulation or deeper into the tumor, these macromolecules would ordinarily be drained to the lymphatic system, which is malfunctioning in inner necrotic tumor tissue. However, these macromolecules can be macropinocytosed into the tumor periphery where they are degraded for nutrition for tumor growth. As the concentration of albumin drops in the vasculature, the liver synthesizes more to maintain the steady-state 0.75 mM systemic concentration. It is thus not surprising to observe that the tumor is where the majority of albumin degradation occurs. Increased vascular permeability is also commonly observed in various inflammatory diseases. As such, EPR-mediated accumulation of albumin and IgG in pathologic sites can be utilized.

Albumin and IgG are attractive drug carriers not only because drug molecules latched on these proteins can enjoy a long circulatory life but also because there exists a strong possibility that drug bound to these proteins can be delivered to solid tumor. There are at least two ways to exploit albumin as a drug carrier. One is direct chemical conjugation of drug molecules to the protein. The second approach is derivatizing a drug with a molecule that increases the drug's affinity for albumin.

The binding affinity in the case of fatty acids to albumin originates from two sources: one is entropy (AS)-driven hydrophobic interaction between the long alkyl chain of fatty acid and the binding cavity and the other is enthalpy (AH)-driven electrostatic attraction between the carboxylic acid anion and positive charges from Lys and Arg in the periphery of the binding pocket. Because of the high affinity of fatty acids toward albumin, a variety of drugs have been modified with fatty acids (Lambert, 2000). However, in all cases, the -COOH group is directly attached to drug molecules because chemistries involved are often straightforward. An important drawback in this simple approach is that the conjugate will bind albumin with a lower affinity than free fatty acids because the electrostatic contribution is no longer available in the interaction of the conjugate with fatty acid binding site. The technology described herein involves both enthalpy as well as entropy contributions to albumin bind of the conjugate.

The neutral pH of the serum causes dissociation and the intact proteins return to the circulation. This protection manifests in an astounding characteristic for these macromolecules: the half-lives of albumin and IgG are 19 and 21 days, respectively. In summary, albumin is produced in the liver as the most abundant protein in the serum. It binds fatty acids with very high affinity and capacity, circulates for a useful length of time and passively targets developing tumors. The above half-life for albumin is in a non-tumor bearing state. Lipids with high affinity dissociate from albumin slowly while low affinity lipids dissociate at a higher rate. Thus the affinity of the conjugate for albumin can be modulated to generate a drug-specific controlled release

formulation.

Different formulations will require conjugates of different affinities which can be modulated through several mechanisms. It is likely that affinity will decrease as the tail length of the fatty acid decreases and the affinity will increase as the tail length increases up to about 22 carbons. Longer chain carbon tails can still be useful. The affinity also appears dependent on the presence of an anionic charge at the head of the molecule putatively due to electrostatic interactions with lysines and arginines on the surface of the binding pocket on albumin. Removal of the charge altogether will decrease affinity while increasing the number of charges or the flexibility of the arm bearing the charge could increase the affinity. The flexibility of the molecule can influence affinity, for example, the flexibility of the 3 carbon symmetrical head group, malonic acid, can be different than the more flexible 5 carbon symmetrical head group, glutaric acid. However, each of these allows maintenance of one anionic charge. The more flexibility to orient the charge at the surface of the pocket can increase affinity. Similarly, a 4 carbon asymmetrical head group, such as succinic acid, is useful. The succinic acid can also be cyclized into an anhydride allowing facile conjugation with nucleophiles. A succinic acid based lipid as well as any other alkyldioic acid could be used as a head group for the formulation of lipid drug conjugates for the purpose of binding albumin. By alkyldioic acid is meant a straight alkyl or alkenyl chain with carboxylates at the distal ends which include; malonic acid, succinic acid, maleic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid and the like.

The long-chain fatty acid-SERD conjugate compound formulations for cancer can be used particularly in the treatment of solid tumors. "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The term "solid tumor" refers to a cancer or carcinoma of body tissues other than blood, bone marrow, and lymphoid system. The compounds and methods described herein are for treating human subjects with solid tumors, specifically, tumors comprising tamoxifen-resistant tumor cells. In particular, this refers to a particularly recalcitrant form of metastatic breast cancer.

In instances where the compounds described herein possess one or more stereocenters, each stereocenter exists independently in either the R or S configuration. The compounds presented herein include all diastereomeric, enantiomeric,

atropisomers, and epimeric forms as well as the appropriate mixtures thereof. The compounds and methods provided herein include all cis, trans, syn, anti, entgegen (E), and zusammen (Z) isomers as well as the appropriate mixtures thereof. The chemical structures depicted herein are intended include both (E) and (Z) isomers unless specifically described as a single isomer. Stereoisomers are obtained, if desired, by methods such as, stereoselective synthesis and/or the separation of stereoisomers by chiral chromatographic columns and/or use of optically active resolving agents. In certain embodiments, the compounds presented herein are present as atropisomers. Atropisomers refer to stereoisomers resulting from hindered rotation about single bonds where the steric strain barrier to rotation allows for the isolation of conformers.

Atropisomers display axial chirality. Separation of atropisomers is possible. In some embodiments, separation of atropisomers is possible by chiral resolution methods such as selective crystallization. Atropisomers are optionally characterized by NMR or other suitable characterization means.

The methods and compositions described herein include the use of amorphous forms as well as crystalline forms (also known as polymorphs). In one aspect, compounds described herein are in the form of pharmaceutically acceptable salts. As well, active metabolites of these compounds having the same type of activity are included in the scope of the present disclosure. In addition, the compounds described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the compounds presented herein are also considered to be disclosed herein.

In some embodiments, compounds described herein are prepared as prodrugs. A "prodrug" refers to an agent that is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they are easier to administer than the parent drug or they are bioavailable by oral administration or they have improved solubility in pharmaceutical compositions over the parent drug. In some embodiments, the design of a prodrug increases the effective water solubility. An example, without limitation, of a prodrug is a compound described herein, which is administered as an ester (the "prodrug") but then is metabolically hydrolyzed to provide the active entity. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound.

The compound can be formulated as a solution, suspension, suppository, tablet, granules, powder, capsules, ointment, or cream. A variety of additives can be added to these formulations, such as a solvent (e.g., water or physiological saline), solubilizing agent (e.g., ethanol, Polysorbates, or Cremophor EL®), agent for achieving isotonicity, preservative, antioxidizing agent, excipient (e.g., lactose, starch, crystalline cellulose, mannitol, maltose, calcium hydrogen phosphate, light silicic acid anhydride, or calcium carbonate), binder (e.g., starch, polyvinylpyrrolidone, hydroxypropyl cellulose, ethyl cellulose, carboxy methyl cellulose, or gum arabic), lubricant (e.g., magnesium stearate, talc, or hardened oils), or stabilizer (e.g., lactose, mannitol, maltose, polysorbates, macrogels, or polyoxyethylene hardened castor oils). If suitable, the following compounds can also be added: glycerin, dimethylacetamide, sodium lactate, a surfactant, or a basic substance such as sodium hydroxide, ethylenediamine, ethanolamine, sodium bicarbonate, arginine, meglumine, or trisaminomethane. As discussed above, organic solvents (e.g., ethanol) are not required for pharmaceutical formulations containing compounds of Formula II. However, the solubilizing agents and organic materials listed above can be used if a hydrophobic material (e.g., a second analgesic) is included in the formulation, or if the pharmacokinetic characteristic of the formulation is to be modulated. Pharmaceutical preparations such as solutions, emulsions, tablets, granules, or capsules can be formed with these components or the like. As used herein the term "treating" is intended to refer to all processes wherein there may be a slowing, interrupting, arresting, or stopping of the progression of the cancer or symptoms and does not necessarily indicate a total elimination of the underlying condition.

The dose of the compound of the present invention is determined in

consideration of the cancer to be treated, the overall health of the mammal to be treated, etc.

A therapeutically effective amount of the compositions of the present invention will generally mean administration of from about 0.5 mg/kg to about 500 mg/kg (weight of active compound/body weight of mammal). Preferably the amount is from about 5 mg/kg to about 50 mg/kg administered intravenously q2 weeks or qmonth or a daily oral administration. However, an effective amount may vary from mammal to mammal and can easily be adjusted by one of ordinary skill by varying the volume and frequency of administrations.

The amount of composition administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of

administration, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular composition, and the like. Determination of an effective dosage is well within the capabilities of those skilled in the art coupled with the general and specific examples disclosed herein. The administration can occur in a single administration, but can also occur over several administrations. The benefit of the compounds described herein is bioavailability and prolonged half-life.

Accordingly, fewer administrations or more convenient administrations of the active compound are possible.

Practice of the method of the present invention comprises administering to a subject a therapeutically effective amount of a composition in any suitable systemic or local formulation, in an amount effective to deliver a dosage. In practicing the method of treatment or use of the present methods, a therapeutically effective amount of a composition is administered to a mammal either alone or in combination with other therapies.

For intravenous or parenteral administration, i.e., injection or infusion, the composition may also contain suitable pharmaceutical diluents and carriers, such as water, saline, dextrose solutions, fructose solutions, serum albumin, ethanol, or oils of animal, vegetative, or synthetic origin. It may also contain preservatives, and buffers as are known in the art. When a therapeutically effective amount is administered by intravenous, cutaneous or subcutaneous injection, the solution can also contain components to adjust pH, isotonicity, stability, and the like, all of which is within the skill in the art. A composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to compound an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection Citrate Buffer pH 5.5, or other carriers, diluents and additives as known in the art.

The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additive known to those of skill in the art. Compositions for intravenous or parenteral administration can comprise a suitable sterile solvent, which may be an isotonic aqueous buffer or pharmaceutically acceptable organic solvent. Where necessary, the compositions can also include a solubilizing agent, although the novel compounds described herein are particularly beneficial in that they are amenable to formulating. Compositions for intravenous or parenteral administration can optionally include a local anesthetic to lessen pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form in a hermetically sealed container such as an ampoule or sachette. The pharmaceutical compositions for administration by injection or infusion can be dispensed, for example, with an infusion bottle containing, for example, sterile pharmaceutical grade water or saline. Where the pharmaceutical compositions are administered by injection, an ampoule of sterile water for injection, saline, or other solvent such as a pharmaceutically acceptable organic solvent can be provided so that the ingredients can be mixed prior to administration.

Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection. Useful injectable preparations include sterile suspensions, solutions or emulsions of the active compound(s) in aqueous or oily vehicles. The compositions also can contain solubilizing agents, formulating agents, such as suspending, stabilizing and/or dispersing agent. The formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, and can contain added preservatives. For prophylactic administration, the compound can be administered to a patient at risk of developing one of the previously described conditions or diseases. Alternatively, prophylactic administration can be applied to avoid the onset of symptoms in a patient suffering from or formally diagnosed with the underlying condition.

Oral administration of a composition can be accomplished using dosage forms including but not limited to capsules, caplets, solutions, suspensions and/or syrups. Such dosage forms are prepared using conventional methods known to those in the field of pharmaceutical formulation and described in the pertinent texts, e.g., in Remington: The Science and Practice of Pharmacy (2000), supra.

The dosage form may be a capsule, in which case the active agent-containing composition may be encapsulated in the form of a liquid. Suitable capsules may be either hard or soft, and are generally made of gelatin, starch, or a cellulosic material, with gelatin capsules preferred. Two-piece hard gelatin capsules are preferably sealed, such as with gelatin bands or the like. See, for e.g., Remington: The Science and Practice of Pharmacy (2000), supra, which describes materials and methods for preparing encapsulated pharmaceuticals.

Capsules may, if desired, be coated so as to provide for delayed release.

Dosage forms with delayed release coatings may be manufactured using standard coating procedures and equipment. Such procedures are known to those skilled in the art and described in the pertinent texts (see, for e.g., Remington: The Science and Practice of Pharmacy (2000), supra). Generally, after preparation of the capsule, a delayed release coating composition is applied using a coating pan, an airless spray technique, fluidized bed coating equipment, or the like. Delayed release coating compositions comprise a polymeric material, e.g., cellulose butyrate phthalate, cellulose hydrogen phthalate, cellulose proprionate phthalate, polyvinyl acetate phthalate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate, dioxypropyl methylcellulose succinate, carboxymethyl ethylcellulose, hydroxypropyl

methylcellulose acetate succinate, polymers and copolymers formed from acrylic acid, methacrylic acid, and/or esters thereof.

Sustained-release dosage forms provide for drug release over an extended time period, and may or may not be delayed release. Generally, as will be appreciated by those of ordinary skill in the art, sustained-release dosage forms are formulated by dispersing a drug within a matrix of a gradually bioerodible (hydrolyzable) material such as an insoluble plastic, a hydrophilic polymer, or a fatty compound. Insoluble plastic matrices may be comprised of, for example, polyvinyl chloride or polyethylene. Hydrophilic polymers useful for providing a sustained release coating or matrix cellulosic polymers include, without limitation: cellulosic polymers such as

hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethyl cellulose phthalate, hydroxypropylcellulose phthalate, cellulose hexahydrophthalate, cellulose acetate hexahydrophthalate, and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, acrylic acid alkyl esters, methacrylic acid alkyl esters, and the like, e.g. copolymers of acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, with a terpolymer of ethyl acrylate, methyl methacrylate and trimethylammonioethyl methacrylate chloride (sold under the tradename Eudragit RS) preferred; vinyl polymers and copolymers such as polyvinyl pyrrolidone, polyvinyl acetate,

polyvinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate copolymers; zein; and shellac, ammoniated shellac, shellac-acetyl alcohol, and shellac n-butyl stearate. Fatty compounds for use as a sustained release matrix material include, but are not limited to, waxes generally (e.g., carnauba wax) and glyceryl tristearate.

The compounds described herein include all salt forms thereof. Examples of such salts include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, butyrate, citrate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Salts derived from

appropriate bases include alkali metal (e.g., sodium, potassium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄ ⁺ salts, or amino acids such as lysine, arginine, aspartic acid or glutamic acid. Compounds of the formulae herein include those having quaternization of any basic nitrogen-containing group therein. The compounds of this invention may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included in the present invention.

Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds. The term "stable", as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein, e.g., therapeutic administration or storage until use).

The subject matter will now be further described by way of the following non- limiting examples.

EXAMPLES

Example 1

SYNTHESES

Synthesis of NL3. This compound was synthesized according to published methods. (Weatherman RV, Clegg NJ, Scanlan TS. (2001). Chemistry and Biology 8:427-436).

Synthesis of NL4. This compound was synthesized according to published methods (Weatherman RV, Clegg NJ, Scanlan TS. (2001). Chemistry and Biology 8:427-436).

Synthesis of PDG anhydride. This compound was synthesized according to published methods (Hackett MJ et al. (2012). J. Pharm. Sci. doi: 10.1002/jps.23213)

Synthesis of NL4-PDG. To a solution of 100 mg (0.267 mmol, 1 eq) NL4 and 88 mg (0.267 mmol, leq) PDG anhydride in 5 mL pyridine was added 1 14.2 mg (0.935 mmol, 3.5 eq) p-dimethylaminopyridine and solution was allowed to react at RT for 48h. It was observed that the reaction proceeded to about 50% yield. The volatiles were removed under vacuum and the mixture taken up in methyl t-butylether and washed with IN HC1. The organic layer was dried over MgS0₄, filtered and concentrated. The mixture was dissolved in MeOH and eluted on a Zorbax stablebond CI 8 reverse phase column at 49: 1 MeCN: l% AcOH(aq). The product was pooled and concentrated with toluene to remove excess AcOH leaving a slightly yellow solid, 84.67 mg (46% yield).

Synthesis of NL3m. This compound was received as the HC1 salt. Briefly NL3 was activated by isobutylchloroformate (IBCF) for 30 minutes at 4°C in the presence of N-methylmorpholine (NMM). After activation the NL3 was added drop wise to a solution of excess ethylenediamine at 4°C. When the reaction was complete the solution was concentrated under vacuum and the NL3m was precipitated from an organic solution as an HC1 salt.

Synthesis of NL3m-PDG. To a suspension of 667 mg (1.54 mmol, leq) NL3m in 50 mL 4: 1 DCM:pyridine was added 0.86 mL (6.16 mmol, 4eq) triethylamine and the mixture stirred at RT. A solution of 500 mg (1.54 mmol, leq) PDG anhydride was dissolved in 10 mL pyridine and added to the suspension which immediately became a solution. The reaction mixture stirred overnight at RT then was concentrated and the mixture was dissolved in MeOH and injected on a Zorbax stablebond CI 8 reverse phase preparative column with a 93/7 MeCN/l%AcOH(aq) eluent. The product was pooled and suspended in toluene to evaporate excess AcOH leaving a white solid, 0.937 g (84%).

Synthesis of NL3eg-PDG. As with the synthesis of NL3m-PDG, NL3 is activated by IBCF in THF for 30 min at 4°C in the presence of NMM. The activated NL3 is then added dropwise to a solution of excess ethylene glycol and warmed to room temperature. The volatiles are removed under vacuum and the mixture taken up in Et₂0 and washed multiple times with water. The Et₂0 layer is dried over MgS0₄, filtered and concentrated.

Synthesis of paba-PDG. A slight excess of p-aminobenzyl alcohol (paba) is dissolved in DCM with PDG anhydride and the solution is refluxed until all the PDG anhydride is consumed. The reaction mixture is then extracted against IN HC1 to remove paba and the DCM layer is dried over MgS0₄, filtered and concentrated.

NL3paba-PDG. NL3 is activated for 30 minutes at 4°C in THF with IBCF in the presence of NMM then the mixed anhydride is added dropwise to a solution of 1 equivalent of paba-PDG in THF at 4°C. The reaction mixture is allowed to warm to room temperature and react until both materials are consumed. The reaction mixture is concentrated under vacuum, dissolved in MeOH and purified on a CI 8 reverse phase column.

Example 2

In vivo Study in Tamoxifen-Na^'fve and -Resistant Tumors Orthotopic xenograft mouse models were generated to determine efficacy of NL4-PDG for preventing tumor growth compared to currently approved medications.

Results: In the tamoxifen-nai^'ve tumors, NL4-PDG was better than the current approved medication fulvestrant (which was administered at ~140x the approved human dose) but not as good as the gold-standard, tamoxifen. In the tamoxifen- resistant tumors, intravenous NL4-PDG was lOx more potent than fulvestrant and subcutaneously administered NL4-PDG or NL4.

The study was performed in tamoxifen-nai^'ve MCF7 human xenograft tumors orthotopically inoculated in the mammary fat pad. These tumors are estrogen dependent so a controlled release estrogen tablet was injected subcutaneously to promote tumor growth. Studies began when the tumors reached 0.2cm³ in volume. Mice were treated with 1 mg/mouse of tamoxifen (2.69μηιο1Λ1θ86;8.07μηιο1Λνβ6^, NL3 (2.82μηιο1 θ86;8.46μηιο1Λν66ΐ-), NL3m (2.31 μmol/dose;6.93μmol/week) and

(23 ^mol/dose;6.93^ol/week) and NL4-PDG (2J0^ol/dose;8J0^ol/week) three times weekly.

Due to the drastic insolubility of fulvestrant, it is formulated in corn oil and administered 5mg weekly as a single intramuscular injection

The MTD for humans is 500mg / month. Using the standard masses of 70kg for a human and 20g for a mouse, this accounts for ~ 140-fold increase in drug exposure/mass in mice compared to the maximum human dose. While this dose is clinically irrelevant, it is used to show efficacy in preclinical models.

NL3m showed irritation upon injection and had to be cancelled. NL3m-PDG improved the tolerability of the drug and the mice were allowed to proceed through the study. NL4 and NL4-PDG also showed minor irritation upon subcutaneous administration over time.

The results showed cytostatic activity for NL4-PDG, NL4, NL3 and fulvestrant based on increased tumor doubling time and time to 0.7cm³ tumor volume. The results can be summarized as tamoxifen > NL4-PDG > NL4 > NL3 > fulvestrant. NL3m-PDG did not show activity. See Figure 1 A & B.

The tamoxifen-resistant efficacy study involved generating tamoxifen resistant MCF7 tumors in vivo and implanting them into mice orthotopically in the mammary fat pad. In this scenario, tamoxifen had become an agonist for ER activity and was thus used in all formulations as a stimulant for tumor growth, just as estrogen was used in the tamoxifen-nai^'ve study.

To overcome the adverse effects of the subcutaneous administrations, an IV formulation of NL4-PDG was examined. This formulation was pre-incubated with murine serum albumin to solubilize and stabilize the drug making it suitable for IV injection in contrast to all other molecules; this beneficial property is PDG dependent. The amount of albumin administered, 30 mg/mL, provided enough drug binding sites to accommodate 1/10th of the subcutaneous dose

Despite this dose-reduction, the IV formulation was the most stable and biocompatible and was found to be equally effective as the 5mg weekly administration of fulvestrant which was dosed at -140 x the MTD for humans. The NL4, NL4-PDG and NL3 subcutaneous formulations all showed efficacy in this model considering the same endpoints as the first study; tumor doubling time and time to 0.5cm³ tumor volume. The results can be summarized as fulvestrant = NL4-PDG IV (O.lx dose) > NL4-PDG SC > NL3 > NL4. Thus, NL4-PDG is significantly and unexpectedly superior to NL-4 and fulvestrant in efficacy. See Figures 2A & B. The P values from Kaplan-Meir comparisons (Logrank Chia square) are indicated in Table 1.

Table 1.

Example 3

Study of Impedance of Estrogen-Dependent Tumor Growth A study was undertaken utilizing the well-established MCF7 xenograft model of estrogen dependent breast tumors. Tumor growth was the primary endpoint. 95 female athymic nude/nude mice (6 weeks of age) were ovariectomized with simultaneous implantation of a 0.72 mg estradiol pellet designed to release estradiol at a consistent rate over 60 days. 48 hours later, mice bearing terminal MCF7 xenograft tumors (approx. 1cm tumor volume) were sacrificed prior to sterile excision and dicing of the parental tumors. Small (2mm x 2mm) fragments taken from 5 donor tumors were inserted via trochar into an axillary mammary fat pad (1 fragment per mouse) of the prepared 95 mice. Tumor growth and animal weight were monitored 3 times per week until tumors attained 0.2cm3 volume, at which point mice were randomized to one of 9 treatments: 1) Control (vehicle); 2) tamoxifen; 3) fulvestrant; 4) NL3; 5) NL3m; 6) NL4; 7) NL3m-PDG; 8) NL4-PDG and 9) Estrogen withdrawal (vehicle).

Ten-twelve animals were randomized to groups 1-8 (with the exception of group 5 - see "treatment related events" below), and 5 animals to group 9. Treatments were administered 3 x weekly by subcutaneous injection in a volume of 0.1-0.2cc for a dose of lmg or lmg equivalent (PDG compounds) per mouse. The exception was fulvestrant, where 5mg administered 1 x weekly by subcutaneous injection. Tumor size and animal weight were monitored 3 x weekly until tumors achieved >0.9cm³ or 42 days after tumor implantation, at which point the animals were sacrificed and samples taken.

Data Analysis: Mean volume ± SEM for the remaining tumors at each timepoint were plotted up to 21 days post-treatment (Figure 1 A). Data was fit to an exponential growth regression model [Y=Start*exp(K*X), where we constrained the Start to being shared between all groups and doubling time = 0.69/K]. The R² values and doubling times from this analysis are indicated in Table 2.

Table 2. Statistical analysis of Tumor Growth. R² and tumor doubling times as determined by exponential growth curve fits to data. Kaplan-Meir analysis derived P- values comparing indicated group to that of E2-control. Treatment E2 fulvestran tamoxife NL3 NL3m- NL4 NL4- E2 t n PDG PDG Withdrawa

1

R² of 0.546 0.5393 0.2176 0.593 0.578 0.403 0.355 -6.791 exponentia 7 1 1 4

1 growth

curve fit

Doubling 10.66 12.86 26.65 15.25 12.1 1 16.21 16.6 6.93E+06 time (days)

Kaplan- P = P = P = P = P= P= P= Meir: time 0.1768 0.0002 0.012 0.285 0.013 0.002 0.0304 to reach 7 9 2 6

0.7cm³

Runs tests were used to determine whether the curves deviated from the model- fit. Curves for NL3 and NL4 were found to deviate significantly from the model. Kaplan-Meir curves (Figure IB) were plotted by using an endpoint of tumors reaching 0.7cm . Kaplan-Meir curves comparing treatment groups to that of E2 control were analyzed by the Logrank Chi-square test and the associated P-values are indicated in Table 2. Tumor growth from each group was also compared directly to that of E2 control and included in Figures 3-15. The P values indicated in the growth curves in these figures are derived from an F-test comparing fit-K values. Doubling times in these figures were determined from curve fits for each tumor plotted up to 31 days post-treatment, and are plotted as Mean ± SEM. A 2-tailed unpaired Student's T-test was performed to yield the indicated P values. Correlation analysis between uterine wet weight (when data was available) and tumor doubling times is displayed in Figures 16- 18. Doubling times for these graphs were determined from curve fits for each tumor plotted up to 31 days post-treatment. Uterine wet weights are a ratio of uterine weight : body weight. Both axes were Ln transformed prior to linear regression. Runs tests were used to determine whether lines departed from the linear model. The indicated P value represents the probability that the slope of the line is equal to 0 (as determined by F- test).

Results: Tumors of mice within the vehicle control (estradiol only) exhibited growth rates that compare favorably with historical data. Estradiol withdrawal completely suspended, while tamoxifen significantly reduced, tumor growth rate.

Tumor growth was also retarded in mice treated with either of the antiestrogens fulvestrant, NL3 or NL4 compared to those treated with estradiol. NL3m-PDG did not significantly affect tumor growth in this study.

However, NL4-PDG treatment significantly increased tumor doubling time (16.6 vs. 10.66d) and increased time to 0.7cm compared to estradiol treated (Figure 1). In this analysis, the overall impact of antiestrogens on tumor growth was: tamoxifen > NL4-PDG > NL4 > NL3 > fulvestrant > NL3m-PDG.

In this experiment, fulvestrant was not as efficacious as expected. One possible reason was due to the bioavailability of fulvestrant. Therefore, we correlated a well- established biomarker of E2 exposure (uterine wet- weight) to tumor doubling time (Figures 16-18). It is clear that mice with smaller uteri, indicating an increased fulvestrant exposure, had smaller tumor doubling times (r²=0.5368, slope = -1.072, P=0.0248). This correlation did not hold for any of the other compounds. Therefore, we conclude that the poor efficacy of fulvestrant in this study is likely due to a lack of tumor exposure.

Treatment related events: While both PDG formulations were generally well tolerated, the animals treated with NL3m (modified NL3 to allow attachment of the PDG moiety) exhibited significant inflammation at the injection sites. Severe swelling of the flanks after a single administration was noted, and the mice randomized to this treatment (N=2) were euthanized after two administrations. After chronic

administration of NL3m-PDG, we noted the formation of fibrous plaques in the skin and or subcutaneous regions localized around the injection sites in several mice.

However, swelling and inflammation did not appear to be present. Lesions forming at the injection sites of mice treated with either NL4-PDG or NL4 were noted in most mice. These were treated with antibiotic ointment.

All documents cited or referenced in the application cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. A compound having a structure of Formula I:

wherein,

A is a residue of a SERD,

p is from 0 to 500,

L is a linker,

R¹ is a C_8-25 alkyl,

R is a hydroxyl or Ci_-4 alkoxy, and

x and y are each independently zero, one or two,

provided that if L is other than ethylene glycol, then p is one or zero.

2. The compound of claim 1 , wherein one of x and y is one, the other of x and y is zero and z is a double bond.

3. The compound of claim 1 , wherein x and y are both one, wherein one of is a double bond or both of are single bonds.

4. The compound of claim 3, having a structure of Formulae la:

wherein, A is a residue of a SERD,

p is from 0 to 500,

L is a linker,

R¹ is a C_8-25 alkyl, and

R² is a hydroxyl or Ci_-4 alkoxy,

provided that if L is other than ethylene glycol, then p is one or zero.

5. The compound of claim 1 , having a structure of Formulae F or la':

wherein,

A is a residue of a SERD,

p is from 0 to 500,

L is a linker,

R¹ is a C_8-25 alkyl,

R² is a hydroxyl or C alkoxy, and

x and y are each independently zero, one or two,

provided that if L is other than ethylene glycol, then p is one or zero.

6. The compound of claim 1, wherein R is a Cio_-20 alkyl.

7. The compound of claim 1, wherein R¹ is a C12-16 alkyl.

8. The compound of claim 1, wherein R is Ci₅ alkyl.

9. The compound of claim 1, wherein L is selected from the group consisting of

-O O- \ / , (ethylene glycol)

f-NH HN-

, and

111

-O HN-

IV

10. The compound of 9, wherein said SERD is NL4.

1 1. The compound of claim 1, wherein p is zero.

12. The compound of claim 11, having a structure of the formula:

13. The compound of claim 12, wherein R¹ is a C 12-16 alkyl.

14. The compound of claim 12, wherein R is a methoxy or ethoxy.

15. The compound of claim 1 1, having the structure:

16. The compound of claim 1, wherein p is one.

17. The compound of claim 9, wherein L is ethylene glycol and p is from one to 250.

18. The compound of claim 17, wherein p is from one to 50.

19. The compound of claim 17, wherein p is from one to 15.

20. The compound of claim 16, having a structure of formulae:

IV or

wherein, R is hydroxyl.

21. The compound of claim 20, wherein R² is hydroxyl and R¹ is a C]_2-i₆ alkyl.

22. The compound of claim 20, wherein L is selected from the group consisting of

ethylene glycol

-NH HN-

, and

111

-O HN-i

\ / ⁵ IV

23. A pharmaceutical composition comprising a compound of claim 1 and pharmaceutically acceptable excipients and diluents.

24. A method of treating cancer comprising administering a composition of claim 23 to a mammal.

25. The method of claim 24, wherein said cancer is tamoxifen-resistant.