# Patent

## Title
Heterobifunctional inhibitors of E-selectins and CXCR4 chemokine receptors

## Abstract

Compounds, compositions and methods are provided for treating cancer and inflammatory diseases, and for releasing cells such as stem cells (e.g., bone marrow progenitor cells) into circulating blood and enhancing retention of the cells in the blood. More specifically, heterobifunctional compounds that inhibit both E-selectins and CXCR4 chemokine receptors are described.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61/174,580 filed May 1, 2009, which application is incorporated herein by reference in its entirety.


BACKGROUND
1. Technical Field
The present invention relates generally to compounds, compositions and methods for treating cancer and inflammatory diseases, and for enhancing retention of cells after releasing into circulating blood. More specifically, the present invention relates to heterobifunctional compounds that inhibit E-selectins and CXCR4 chemokine receptors, and uses thereof.
2. Description of the Related Art
A number of cancers are highly treatable when treated before the cancer has moved beyond the primary site. However, often once the cancer has spread beyond the primary site, the treatment options are limited and the survival statistics decline dramatically. Bones are a common location for cancer to infiltrate once leaving the primary tumor location. Breast and prostate cancer are examples of cancers that migrate to bones. Even leukemic cells that arise in the bloodstream may home to the bone marrow. Once cancer resides in bone, it is frequently a cause of pain to the individual. Further, once in the bone marrow, the cancer cells may also become resistant to chemotherapy. In addition, if the particular bone affected is a source for production of blood cells in the bone marrow, the individual may develop a variety of blood cell related disorders. Thus, it is desirable to prevent cancer cells from leaving the primary site, or to prevent extravasation of cancer cells from the bloodstream and infiltration into other tissues. Retention of cancer cells in the bloodstream makes the cells more susceptible to treatment, such as chemotherapy.
Some cancers originate all or in part in bone. For such cancers, it is desirable to mobilize cancer cells from bone to the bloodstream and to prevent those cells (as well as any cancer cells already in the bloodstream) from homing to bone or otherwise leaving the bloodstream. Retention of cancer cells in the bloodstream (or mobilization of cancer cells into the bloodstream and then retention therein) makes the cells more susceptible to treatment, such as chemotherapy.
Hematopoietic stem cells (HSCs) also reside in the bone marrow and are a source of material for cellular therapy. HSCs adhere to the stroma within the bone marrow and in order to be harvested must break these adhesions and mobilize out of the bone marrow. It is desirable to have improved agents to increase the HSCs available for harvesting. Such HSCs are useful for engraftment.
Accordingly, there is a need in the art for the treatment of cancers that may leave the primary site and cancers that originate all or in part in bone, and for improved methods to aid in the preparation of therapeutic-grade stem cells. The present invention fulfills these needs and further provides other related advantages.
BRIEF SUMMARY
Briefly stated, compounds, compositions and methods for treating diseases and for improving methods in which an E-selectin and a CXCR4 chemokine receptor play a role, are provided. In the present invention, the compounds are heterobifunctional compounds wherein an E-selectin inhibitor is linked to a CXCR4 chemokine receptor inhibitor. Such compounds may be combined with a pharmaceutically acceptable carrier or diluent to form a pharmaceutical composition. The compounds may be used to treat cancer in which the cancer cells may leave the primary site, or to treat an inflammatory disease in which the adhesion or migration of cells occurs in the disease, or to release cells such as stem cells (e.g., bone marrow progenitor cells) into circulating blood and enhance retention of the cells in the blood (e.g., to mobilize cells out of bone marrow and maintain the cells in the peripheral bloodstream).
The present invention provides a heterobifunctional compound for inhibition of E-selectin and the CXCR4 chemokine receptor, comprising E-selectin inhibitor-Linker-CXCR4 chemokine receptor inhibitor, or a physiologically acceptable salt thereof.
In one embodiment of the compound, the E-selectin inhibitor consists of:




wherein:

    
    
        L=end of bond to Linker;
        R1═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, OH, or NHX where X═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, or OH; C(═O)OX, alkanyl substituted with C(═O)OX, C(═O)NHX, alkanyl substituted with C(═O)NHX, where X═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, or OH; C(═O)X, OX, NHX, NHC(═O)X, where X═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, or OH;
        R2═—OH,
    
    






—O—C(═O)—X, —NH2, —NH—C(═O)—NHX, or —NH—C(═O)—X where n=0-2 and X is independently selected from C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl,





where Q is H or a physiologically acceptable salt, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, aryl, (CH2)m-aryl where m is 1-10, and where n=0-10, and any of the above ring compounds may be substituted with one to three independently selected of Cl, F, CF3, C1-C8 alkoxy, NO2, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, C1-C14 aryl, or OY, C(═O)OY, NY2 or C(═O)NHY where Y is H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, or C1-C14 aryl;

    
    
        R3═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, CN, CH2CN, C(═O)X where X is H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, NHOH, NHOCH3, NHCN, or NX2, or C(═O)OY where Y is H, C1-C8 alkanyl, C1-C8 alkenyl or C1-C8 alkynyl; and
    
    






    
    
        
        
            where the cyclopropane ring may be substituted with one to two, and the cyclohexane ring may be substituted with one to three, independently selected of Cl, F, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl or OY where Y is H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl or C1-C14 aryl.
        
        
    
    


In one embodiment of the compound, the E-selectin inhibitor consists of:




wherein L=end of bond to Linker.

In one embodiment of the compound, the E-selectin inhibitor consists of:




wherein L=end of bond to Linker.

In one embodiment of the compound, the E-selectin inhibitor consists of:




wherein L=end of bond to Linker.

In one embodiment of the compound, the E-selectin inhibitor consists of:




wherein L=end of bond to Linker.

In one embodiment of the compound, the CXCR4 chemokine receptor inhibitor consists of:




wherein L=end of bond to Linker.

In one embodiment, the compound has the formula:




wherein:

    
    
        R1═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, OH, or NHX where X═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, or OH; C(═O)OX, alkanyl substituted with C(═O)OX, C(═O)NHX, alkanyl substituted with C(═O)NHX, where X═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, or OH; C(═O)X, OX, NHX, NHC(═O)X, where X═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, or OH:
        R2═—OH,
    
    






—O—C(═O)—X, —NH2, —NH—C(═O)—NHX, or —NH—C(═O)—X where n=0-2 and X is independently selected from C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl,





Q is H or a physiologically acceptable salt, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, aryl, (CH2)m-aryl where m is 1-10, and where n=0-10, and any of the above ring compounds may be substituted with one to three independently selected of Cl, F, CF3, C1-C8 alkoxy, NO2, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, C1-C14 aryl, or OY, C(═O)OY, NY2 or C(═O)NHY where Y is H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, or C1-C14 aryl;

    
    
        R3═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, CN, CH2CN, C(═O)X where X is H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, NHOH, NHOCH3, NHCN, or NX2, or C(═O)OY where Y is H, C1-C8 alkanyl, C1-C8 alkenyl or C1-C8 alkynyl; and
    
    






where the cyclopropane ring may be substituted with one to two, and the cyclohexane ring may be substituted with one to three, independently selected of Cl, F, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl or OY where Y is H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl or C1-C14 aryl.

In one embodiment, the compound has the formula:




In one embodiment, the compound has the formula:




In one embodiment, the compound has the formula:




In one embodiment, the compound has the formula:




In one embodiment, the Linker of the compound is —C(═O)—NH—(CH2)2—NH—.
In one embodiment, the Linker of the compound is —CH2—NH—CH2—.
In one embodiment, the Linker of the compound is —C(═O)—NH—CH2—.
These linkers, as well as the others disclosed herein and those otherwise known in the art, are for use in a compound of the present invention such as the four embodiments depicted above containing a Linker.
The present invention provides a method for the treatment of a cancer in which the cancer cells may leave the primary site in an individual who is in need of such treatment, comprising administering to the individual a compound of the present invention in an amount effective for treatment, wherein the compound is with or without a pharmaceutically acceptable carrier or diluent.
The present invention provides a method for the treatment of a cancer in which it is desired to mobilize cancer cells from a site into the bloodstream and retain the cancer cells in the bloodstream in an individual who is in need of such treatment, comprising administering to the individual a compound of the present invention in an amount effective for treatment, wherein the compound is with or without a pharmaceutically acceptable carrier or diluent.
The present invention provides a method for releasing cells into circulating blood and enhancing retention of the cells in the blood of an individual who is need of such treatment, comprising administering to the individual a compound of the present invention in an amount effective for treatment, wherein the compound is with or without a pharmaceutically acceptable carrier or diluent. In an embodiment, the method further includes the step of collecting the cells released. In an embodiment, the step of collecting utilizes apheresis. In an embodiment, the cells are stem cells (e.g., bone marrow progenitor cells). In an embodiment, G-CSF is administered to the individual.
The present invention provides a method for the treatment of an inflammatory disease in which the adhesion or migration of cells occurs in the disease in an individual in need of such treatment, comprising administering to the individual a compound of the present invention in an amount effective for treatment, wherein the compound is with or without a pharmaceutically acceptable carrier or diluent.
The present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable carrier or diluent.
In other embodiments, the above compounds thereof may be used in the manufacture of a medicament, and for any of the uses recited herein.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 (FIG. 1A and FIG. 1B) is a diagram illustrating the synthesis of heterobifunctional Compound #1 (compound 27).
FIG. 2 (FIG. 2A, FIG. 2B and FIG. 2C) is a diagram illustrating the synthesis of heterobifunctional Compound #2 (compound 28).
FIG. 3 depicts the inhibition of binding of anti-CXCR4-PE to SupT1 cells in a dose-dependent manner by heterobifunctional Compound #1.
FIG. 4 depicts the results of an E-selectin assay in which heterobifunctional Compound #1 is used as the inhibitor.
FIG. 5 depicts a comparison of IC50 values of compounds A, B and #1 for E-selectin. Compound A, which is a known E-selectin inhibitor, is compound 15 of Thoma et al. (J. Med. Chem. 42:4909-4913, 1999) and is used as a reference compound. Compound #1 is heterobifunctional Compound #1. Compound B is the glycomimetic portion of Compound #1 (i.e., compound 18 of FIG. 1 except modified to replace COOMe, which is used in the linking process, with H).



DETAILED DESCRIPTION
As noted above, the present invention provides compounds, compositions and methods for treating diseases in which an E-selectin and a CXCR4 chemokine receptor play a role, and for enhancing retention of cells after releasing into circulating blood. The compounds have a variety of uses in vitro and in vivo.
As used herein, the term “E-selectin inhibitor” refers to an inhibitor of E-selectin only, as well as to an inhibitor of E-selectin and either P-selectin or L-selectin, or E-selectin and both P-selectin and L-selectin. Thus, there is E-selectin inhibition regardless of whether there is also inhibition of either P-selectin or L-selectin or both P-selectin and L-selectin.
All compounds of the present invention or useful thereto (e.g., for pharmaceutical compositions or methods of treating) include physiologically acceptable salts thereof. Examples of such salts are Na, K, Li, Mg, Ca, and Cl.
A compound of the present invention is a heterobifunctional compound wherein an E-selectin inhibitor is linked (i.e., covalently bonded) to a CXCR4 chemokine receptor inhibitor. Such a compound comprises, or consists of, the formula:
E-selectin inhibitor-Linker-CXCR4 chemokine receptor inhibitor. Accordingly, the compound functions to inhibit E-selectin and to inhibit the CXCR4 chemokine receptor.
E-selectin inhibitors are well known in the art. Some E-selectin inhibitors are specific for E-selectin only. Other E-selectin inhibitors have the ability to inhibit not only E-selectin but additionally P-selectin or L-selectin or both P-selectin and L-selectin. Examples of E-selectin inhibitors (specific for E-selectin or otherwise) are disclosed in U.S. Pat. No. 7,060,685; U.S. Application Publication No. US-2007-0054870; U.S. Application Publication No. US-2008-0161546; and references cited in any of these patent or published application documents. Those examples are small organic molecules. Other known E-selectin inhibitors are amino acid-based, such as antibodies. For example, the humanized monoclonal antibody CDP850 is an E-selectin inhibitor.
In one embodiment of the compound, the E-selectin inhibitor consists of:




wherein L=end of bond to Linker.

In one embodiment of the compound, the E-selectin inhibitor consists of:




wherein L=end of bond to Linker.

In one embodiment of the compound, the E-selectin inhibitor consists of:




wherein L=end of bond to Linker.

In one embodiment of the compound, the E-selectin inhibitor consists of:




wherein L=end of bond to Linker.

CXCR4 chemokine receptor inhibitors are well known in the art. Such inhibitors will typically prevent the binding of stromal derived factor-1 (SDF-1) to a CXCR4 receptor. Examples of CXCR4 chemokine receptor inhibitors are AMD-3100 (Hendrix et al., Antimicrob. Agents Chemother. 44:1667-1673, 2000); ALX40-4C (Doranz et al., AIDS Research and Human Retroviruses 17:475-486, 2001); and T134 (Arakaki et al., J. Virol. 73:1719-1723, 1999). These examples include a small organic molecule and amino acid-based molecules, such as the T22 peptide. AMD-3100 is a bicyclam. Each of the two cyclam rings is attached to the same phenyl ring (each cyclam ring is para to the other) via a methylene group. In one embodiment of a compound of the present invention, the CXCR4 chemokine receptor inhibitor is a phenyl ring to which is attached only one cyclam ring.
In a compound of the present invention, an E-selectin inhibitor and a CXCR4 chemokine receptor inhibitor are covalently joined via a linker (i.e., interposed between the two inhibitors is a “Linker”). A linker may be (or may include) a spacer group, such as —(CH2)p— or —O(CH2)p— where p is generally about 1-20 (including any whole integer range therein). Other examples of spacer groups include a carbonyl or carbonyl containing group such as an amide. An embodiment of such spacer groups is




Embodiments of linkers include the following:




Other linkers, e.g., polyethylene glycols (PEG) or —C(═O)—NH—(CH2)p—C(═O)—NH2 where p is as defined above, will be familiar to those in the art or in possession of the present disclosure.

In another embodiment, the linker is




In another embodiment, the linker is




In another embodiment, the linker is —C(═O)—NH—(CH2)2—NH—.
In another embodiment, the linker is —CH2—NH—CH2—.
In another embodiment, the linker is —C(═O)—NH—CH2—.
In one embodiment of a compound of the present invention, the E-selectin inhibitor consists of:




wherein L is the end of the bond to Linker.

In the present disclosure, there are several chemical abbreviations. “Me” is methyl. “Et” is ethyl. “Ar” is aryl. “Bz” is benzoyl.
Selection of a substituent at R1 includes H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, OH, or NHX where X═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, or OH; C(═O)OX, alkanyl substituted with C(═O)OX, C(═O)NHX, alkanyl substituted with C(═O)NHX, where X═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, or OH; C(═O)X, OX, NHX, NHC(═O)X, where X═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, or OH.
Selection of a substituent at R2 includes —OH,




—O—C(═O)—X, —NH2, —NH—C(═O)—NHX, or —NH—C(═O)—X where n=0-2 and X is independently selected from C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl,





where Q is H or a physiologically acceptable salt, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, aryl, (CH2)m-aryl where m is 1-10, and where n=0-10, and any of the above ring compounds may be substituted with one to three independently selected of Cl, F, CF3, C1-C8 alkoxy, NO2, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, C1-C14 aryl, or OY, C(═O)OY, NY2 or C(═O)NHY where Y is H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, or C1-C14 aryl.

Selection of a substituent at R3 includes H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, CN, CH2CN, C(═O)X where X is H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, NHOH, NHOCH3, NHCN, or NX2, or C(═O)OY where Y is H, C1-C8 alkanyl, C1-C8 alkenyl or C1-C8 alkynyl; and
Selection of a substituent at R4 includes




where the cyclopropane ring may be substituted with one to two, and the cyclohexane ring may be substituted with one to three, independently selected of Cl, F, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl or OY where Y is H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl or C1-C14 aryl.

As used herein, a “C1-C8 alkanyl” refers to an alkane substituent with one to eight carbon atoms and may be straight chain, branched or cyclic (cycloalkanyl). Examples are methyl, ethyl, propyl, isopropyl, butyl and t-butyl. A “halogenated C1-C8 alkanyl” refers to a “C1-C8 alkanyl” possessing at least one halogen. Where there is more than one halogen present, the halogens present may be the same or different or both (if at least three present). A “C1-C8 alkenyl” refers to an alkene substituent with one to eight carbon atoms, at least one carbon-carbon double bond, and may be straight chain, branched or cyclic (cycloalkenyl). Examples are similar to “C1-C8 alkanyl” examples except possessing at least one carbon-carbon double bond. A “C1-C8 alkynyl” refers to an alkyne substituent with one to eight carbon atoms, at least one carbon-carbon triple bond, and may be straight chain, branched or cyclic (cycloalkynyl). Examples are similar to “C1-C8 alkanyl” examples except possessing at least one carbon-carbon triple bond. An “alkoxy” refers to an oxygen substituent possessing a “C1-C8 alkanyl,” “C1-C8 alkenyl” or “C1-C8 alkynyl.” This is —O-alkyl; for example methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy and the like; and alkenyl or alkynyl variations thereof (except for methoxy). It further refers to the group O-alkyl-W-alkyl where W is O or N; for example —O—(CH2)n—W—(CH2)m where n and m are independently 1-10. An “aryl” refers to an aromatic substituent with one to fourteen carbon atoms as ring atoms in one or multiple rings which may be separated by a bond or fused. As used herein, “aryl” includes “heteroaryl.” A “heteroaryl” is similar to an “aryl” except the aromatic substituent possesses at least one heteroatom (such as N, O or S) in place of a ring carbon. Where an aromatic substituent is an aryl in which all the ring atoms are carbon (i.e., not a heteroaryl), there are typically six to fourteen ring atoms. Where an aryl is a heteroaryl, there may be less than six carbon ring atoms. Examples of aryls include phenyl, naphthyl, pyridinyl, pyrimidinyl, triazolo, furanyl, oxazolyl, thiophenyl, quinolinyl and diphenyl.
In one embodiment of a compound of the present invention, the CXCR4 chemokine receptor inhibitor consists of:




wherein L is the end of the bond to Linker.

In one embodiment, the compound has the formula:




wherein R1, R2, R3 and R4 are as defined above.

In one embodiment in which the linker is —C(═O)—NH—(CH2)2—NH—, the compound has the formula:




wherein R1, R2, R3 and R4 are as defined above.

In one embodiment in which the linker is —CH2—NH—CH2—, the compound has the formula:




wherein R1, R2, R3 and R4 are as defined above.

In one embodiment in which the linker is —C(═O)—NH—CH2—, the compound has the formula:




wherein R1, R2, R3 and R4 are as defined above.

In one embodiment in which the linker is —C(═O)—NH—(CH2)2—NH—, the compound has the formula:




In one embodiment in which the linker is —CH2—NH—CH2—, the compound has the formula:




In one embodiment in which the linker is —C(═O)—NH—CH2—, the compound has the formula:




In one embodiment in which the linker is —C(═O)—NH—(CH2)2—NH—, the compound has the formula:




All compounds of the present invention or useful thereto (e.g., for pharmaceutical compositions or methods of treating), include physiologically acceptable salts thereof. Examples of such salts are Na, K, Li, Mg, Ca and Cl.
Compounds as described herein may be present within a pharmaceutical composition. A pharmaceutical composition comprises one or more compounds in combination with (i.e., not covalently bonded to) one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) or preservatives. Within yet other embodiments, compositions of the present invention may be formulated as a lyophilizate. Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration.
The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration). Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of compound release. The amount of compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
The above-described compounds including equivalents thereof are useful in methods of the present invention. In one embodiment, the compounds may be used in a method for the treatment of a cancer in which the cancer cells may leave the primary site. A primary site may be, for example, solid tissue (e.g., breast or prostate) or the bloodstream. An individual who is in need of such treatment is administered at least one (i.e., one or more) of the above-described compounds in an amount effective for the treatment. In addition to breast cancer and prostate cancer, other examples of infiltrating diseases include lung cancer and melanoma, as well as the hematological malignancies (e.g., leukemias and myelomas). As used herein, the term “treatment” (including variations such as “treating”) includes for the disease or a complication associated with the disease, and includes prevention. For example, a complication associated with the cancer may not have presented itself in an individual with the disease, and a compound may be administered to prevent presentation of the complication in the individual. Complications associated with a cancer in which the cancer cells may leave the primary site include, for example, metastasis and infiltration of cancer cells to other tissues. For example, acute myelogenous leukemia (AML) and multiple myeloma (MM) cells migrate to the endosteal region of the bone marrow where the cells become quiescent and are protected from chemotherapy-induced apoptosis. Administration of a compound described herein may prevent adhesion or migration of cancer cells. Such prevention can result in making the cancer cells more susceptible to treatment with chemotherapy. Administration of a compound described herein in the context of prevention may be to an individual who is at risk of occurrence of a cancer for the first time, or for recurrence of a cancer. For example, while a brain cancer such as glioblastoma multiforme is typically treated with another type of therapy (such as radiation or chemotherapy) for the first occurrence, such therapy is usually not effective to prevent recurrence.
The term “treatment” as used herein refers to any of a variety of positive effects from the treatment including, for example, eradicating a complication associated with the disease, relieving to some extent a complication, slowing or stopping progression of the disease, enhancing the effectiveness of one or more therapies for the disease, and prolonging the survival time of the recipient. The treatment may be used in conjunction with one or more other therapies for a cancer or a complication associated therewith.
In another embodiment, the above-described compounds including equivalents may be used in a method for the treatment of a cancer in which it is desired to mobilize cancer cells from a site into the bloodstream and retain the cancer cells in the bloodstream. An individual who is in need of such treatment is administered at least one (i.e., one or more) of the compounds in an amount effective for the treatment. Examples of cancers for such treatment include leukemias and myelomas (e.g., AML and MM). Mobilizing cancer cells into the bloodstream from a site and retaining the cells therein can result in making the cancer cells more susceptible to treatment with chemotherapy. An example of a site from which to mobilize cancer cells is bone. Cancer cells may, for example, be in circulation and then home to bone. Once in bone, the cancer cells are protected from chemotherapy. A compound described herein may be used, for example, to mobilize cancer cells from bone into the bloodstream and prevent cancer cells from homing to bone, thereby retaining the cancer cells in the bloodstream. Administration of a compound described herein in the context of prevention may be to an individual who is at risk of occurrence of a cancer for the first time, or for recurrence of a cancer. For example, while a brain cancer such as glioblastoma multiforme is typically treated with another type of therapy (such as radiation or chemotherapy) for the first occurrence, such therapy is usually not effective to prevent recurrence.
In another embodiment, the above-described compounds including equivalents may be used in a method for releasing cells (such as hematopoietic stem cells) into circulating blood and enhancing retention of the cells in the blood. An individual who is in need of such treatment is administered at least one (i.e., one or more) of the compounds in an amount effective for the treatment. One use of the method is, for example, for stem cell harvesting. Stem cells may be needed, for example, after high-dose chemotherapy treatment. Many chemotherapies suppress bone marrow which disrupts the production of certain components of blood in an individual. As a result, the individual may develop a variety of blood cell related disorders and continuation of chemotherapy may be compromised. A compound described herein may be used, for example, to release stem cells into circulating blood and enhance retention of the stem cells in the blood. The method may include a further step of collecting cells that are released. For example, released stem cells may be collected. A variety of techniques are known in the art for collecting cells. For example, apheresis may be utilized. An example of a stem cells is a bone marrow progenitor cell. The release of such cells from bone marrow into circulating blood and retention therein has a variety of uses. For example, the mobilized bone marrow progenitor cells may be collected from the blood. A use of such collected cells is to obtain healthy bone marrow progenitor cells from an individual prior to treatment of the individual in a manner such that bone marrow is suppressed. Following treatment, the individual can receive a bone marrow transplantation utilizing the bone marrow progenitor cells collected prior to treatment. This is useful, for example, where an individual needs to be subjected to a chemotherapy protocol that will suppress bone marrow.
It can be desirable to additionally treat an individual with at least one (i.e., one or more) colony stimulating factor. Such a factor may be administered, for example, before or simultaneous with administration of at least one of the above-described compounds. Where administration is simultaneous, the combination may be administered from a single container or two (or more) separate containers. An example of a suitable colony stimulating factor is granulocyte-colony stimulating factor (G-CSF). G-CSF induces the bone marrow to grow and produce more stem cells. A compound described herein aids in releasing stem cells into circulating blood. Stem cells produced in bone marrow and released into circulating blood, as a result of the combination of the administration (separately or together) of a compound described herein and G-CSF, may be collected as described above. Such collected stem cells may be, for example, administered to the individual after chemotherapy. The stem cells return to the bone marrow and produce blood cells. Application of a compound described herein to mobilization and harvesting of healthy bone marrow progenitor cells from bone marrow treated with G-CSF provides cells useful, for example, for bone marrow transplantation.
In another embodiment, the above-described compounds including equivalents may be used in a method for the treatment of an inflammatory disease in which the adhesion or migration of cells occurs in the disease. An individual who is in need of such treatment is administered at least one (i.e., one or more) of the compounds in an amount effective for the treatment. Example of inflammatory diseases include inflammatory skin disorders such as atopic dermatitis and psoriasis. The treatment may reduce (partially or totally) the disease or a complication associated therewith, such as pain. The treatment may be used in conjunction with one or more other therapies for such an inflammatory disease or a complication associated therewith.
The above-described compounds may be administered in a manner appropriate to the disease to be treated. Appropriate dosages and a suitable duration and frequency of administration may be determined by such factors as the condition of the patient, the type and severity of the patient's disease and the method of administration. In general, an appropriate dosage and treatment regimen provides the compound(s) in an amount sufficient to provide therapeutic or prophylactic benefit. Within particularly preferred embodiments of the invention, a compound may be administered at a dosage ranging from 0.001 to 1000 mg/kg body weight (more typically 0.01 to 1000 mg/kg), on a regimen of single or multiple daily doses. Appropriate dosages may generally be determined using experimental models or clinical trials. In general, the use of the minimum dosage that is sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated, which will be familiar to those of ordinary skill in the art.
At least one (i.e., one or more) of the above-described compounds may be administered in combination with at least one (i.e., one or more) agent, e.g., chemotherapeutic agent or anti-inflammatory agent. In addition, the administration may be in conjunction with one or more other therapies for reducing toxicities of chemotherapy. For example, at least one (i.e., one or more) agent to counteract (at least in part) a side effect of chemotherapy may be administered. At least one compound described herein may be administered before, after or simultaneous with administration of at least one chemotherapeutic agent or anti-inflammatory agent. Where administration is simultaneous, the combination may be administered from a single container or two (or more) separate containers.
The following Examples are offered by way of illustration and not by way of limitation.
Examples
Example 1
Synthesis of Heterobifunctional Compound #1 (Compound 27 of FIG. 1)
Synthesis of Compound 2:
Commercially available (Aldrich Chemical Co., Milwaukee, Wis.) cis-1,2,3,6-tetrahydrophthalic anhydride (compound 1, 50 g) is added to a suspension of amberlyste 15 (50 g, dried under vacuum) in methanol (1 L) with stirring. Triethylorthoformate (100 ml) is added immediately while stirring. The reaction mixture is then vigorously stirred for 5 days at room temperature and additional triethylorthoformate is added. Stirring is continued for an additional 4 days, then the reaction mixture filtered over celite and washed with methanol. The solvent is removed in vacuum and the residue is dissolved in CH2Cl2 (200 ml). The solution is washed with a cold saturated solution of NaHCO3 (200 ml) and cold brine (200 ml). The organic layer is dried (Na2SO4), filtered and concentrated to dryness to afford compound 2 (55 g).
Synthesis of Compound 3:
To a suspension of compound 2 (10 g) in phosphate buffer (400 ml, pH 7) is added PLE (40 mg, 1080 unit). The pH of the mixture is maintained at 7 by continuous dropwise addition of 1M NaOH solution via syringe pump. The reaction is stirred at 20° C. until 1 equivalent of NaOH (50 ml) is used. The reaction mixture is transferred to a reparatory funnel and EtOAc (400 ml) is added. The layers are separated and the organic layer is extracted with phosphate buffer (2×250 ml, pH 7). The combined aqueous layers are acidified (pH 2) with aqueous HCl (1M) and extracted with EtOAc (3×400 ml). The combined organic layers are dried (Na2SO4), filtered and concentrated to dryness to afford compound 3 (7.8 g).
Synthesis of Compound 4:
To a solution of compound 3 (2 g) in dry CH2Cl2 (35 ml) is added (COCl)2 (1.4 ml) and DMF (0.025 ml) and stirred for 3 h at RT. The solution is evaporated to dryness (rotavapor is purged with argon). The residue is dissolved in dry THF (40 ml) and is added dropwise over a period of 20 min to a boiling suspension of 2-mercaptopyridine-1-oxide sodium salt (2 g), t-BuSH (6 ml), and 4-DMAP (52 mg) in dry THF (100 ml). The solution is stirred under reflux for 3 h. The reaction mixture is cooled down to RT and transferred into a separatory funnel with EtOAc (100 ml) and washed with H2O (100 ml). The aqueous layer is extracted with EtOAc (2×200 ml). The combined organic layers are dried (Na2SO4), filtered and concentrated to dryness. The crude product is purified by column chromatography (silica) to afford compound 4 as yellowish oil (1.1 g).
Synthesis of Compound 5:
To a suspension of compound 4 (4 g) in phosphate buffer (400 ml, pH 7) is added PLE (42 mg) with stirring. The pH is kept at 7 by adding NaOH solution (1M) via syringe pump. The reaction mixture is stirred at RT until 1 equivalent of NaOH is used. The reaction mixture is transferred to a separatory funnel and washed with EtOAc (2×250 ml). The layers are separated and the organic layers are extracted with phosphate buffer (2×250 ml, pH 7). The combined aqueous layers are acidified to pH 2 with aqueous HCl solution and extracted with EtOAc (3×300 ml). The combined organic layers are dried (Na2SO4), filtered and evaporated to dryness. The crude product is filtered through a short plug of silica to afford compound 5 (3 g).
Synthesis of Compound 6:
Compound 5 (4 g) is suspended in water (90 ml) and cooled down to 0° C. NaHCO3 (8 g) is added followed by a solution of KI (32 g) and I2 (8 g) in water (75 ml). The reaction mixture is stirred at RT for 24 h and then extracted with CH2Cl2 (3×30 ml). The combined organic layers are washed with a saturated solution of Na2S2O3 in water (125 ml). The aqueous layer is extracted with CH2Cl2 (2×30 ml). The combined organic layers are protected from light, dried (Na2SO4), filtered, and concentrated to dryness and quickly under high vacuum to afford iodolactone 6 as an off-white solid (7.5 g).
Synthesis of Compound 7:
Compound 6 (7 g) is dissolved in dry THF (170 ml) and DBU (7 ml) is added. The reaction mixture is refluxed for 20 h and then cooled down to RT. Diethyl ether (100 ml) is added and transferred into a separatory funnel and extracted with an aqueous solution of HCl (200 ml, 0.5M). The aqueous layers are extracted with Et2O (3×100 ml). The combined organic layers are washed with brine (200 ml), dried (Na2SO4), filtered, and concentrated to dryness. The crude product is purified by column chromatography (silica gel) to afford compound 7 (3.7 g).
Synthesis of Compound 8:
NaHCO3 (2.2 g) is dried under vacuum and then dry MeOH (132 ml) is added with stirring followed by compound 7 (3 g). The reaction mixture is then stirred at RT under argon for 12 h. The solvent is evaporated off and the residue is transferred into a separatory funnel with CH2Cl2 (35 ml), extracted with water (40 ml), and with brine (40 ml). The aqueous layer is extracted with CH2Cl2 (2×35 ml). The combined organic layers are dried (Na2SO4), filtered, and concentrated to dryness to give compound 8 (5 g).
Synthesis of Compound 9:
To a solution of compound 8 (4 g) in dry CH2Cl2 (80 ml) is added tert-butyldimethylsilyl chloride (7.2 ml) in small portions, followed by DBU (9.5 ml). The reaction mixture is stirred for 12 h and then quenched with MeOH (12 ml). The reaction mixture is transferred into a separatory funnel with CH2Cl2 (60 ml), washed with cold saturated solution of NaHCO3 (50 ml) and cold brine (50 ml). The aqueous layers are extracted with CH2Cl2 (2×50 ml). The combined organic layers are dried (Na2SO4), filtered and concentrated to dryness. The residue is purified by column chromatography (silica) to give compound 9 (6 g).
Synthesis of Compound 10:
To a cold (10° C.) solution of compound 9 (5 g) in CH2Cl2 (125 ml) is added m-CPBA (8 g) with stirring, and stirring is continued for 15 h at 10° C. The temperature is raised to RT over a period of 2 h and the mixture is diluted with CH2Cl2 (400 ml). The mixture is transferred into a separatory funnel, and washed with cold saturated solution of Na2S2O3 solution in water (2×400 ml). The organic layer is successively washed with cold saturated solution NaHCO3 (400 ml) and cold brine (100 ml). The aqueous layers are extracted with CH2Cl2 (2×400 ml). The combined organic layers are dried (Na2SO4), filtered, and concentrated to dryness. The crude product is purified by column chromatography (silica) to give compound 10 (4 g).
Synthesis of Compound 11:
CuCN (1.5 g) is dried in high vacuum at 150° C. for 30 min, suspended in dry THF (25 ml) and cooled down to −78° C. MeLi (1.6 M in Et2O, 22.5 ml) is added slowly via syringe and the temperature is raised to −10° C. over a period of 30 min. The mixture is again cooled down to −78° C., followed by the addition of BF3 etherate (1.4 ml) in THF (5 ml). After stirring for 20 min, compound 10 (1 g) in THF (25 ml) is added and stirring is continued for 5 h at −78° C. The excess of MeLi is quenched with a mixture of MeOH (10 ml) and Et3N (10 ml). The mixture is diluted with Et2O (250 ml) and transferred into a reparatory funnel and extracted with aqueous 25% NH3/satd. NH4Cl (1:9) solution. The organic layer is successively washed with brine (150 ml), 5% AcOH (150 ml), saturated solution of NaHCO3 (150 ml), and brine (150 ml). The aqueous layers are extracted with Et2O (2×250 ml). The combined organic layers are dried (Na2SO4), filtered, and concentrated to dryness. The crude product is purified by column chromatography (silica) to give compound 11 (800 mg).
Synthesis of Compound 13:
A solution of Br2 (0.08 ml) in CH2Cl2 (1 ml) is added dropwise at 0° C. to a solution of commercially available (Carbosynth Ltd., Compton, Berkshire, UK) compound 12 (640 mg) in CH2Cl2 (10 ml) and stirred at 0° C. for 1 h. Cyclohexene (0.02 ml) is added and the reaction mixture is stirred for anther 30 min. The mixture is added dropwise to a solution of 11 (310 mg) and Et4NBr (280 mg, oven dried at 200° C.) in DMF/CH2Cl2 (20 ml, 1:1) containing molecular sieve (1 g, 3 A) with stirring at RT. The stirring is continued for 60 h. The reaction is quenched with pyridine (2 ml), filtered over celite, and washed with CH2Cl2 (20 ml). The solution is washed with brine (50 ml) and the aqueous layer is extracted 3 times with CH2Cl2 (3×50 ml). The combined organic layers are dried (Na2SO4), filtered, and concentrated to dryness. The crude product is purified by column chromatography (silica) to give compound 13 (144 mg).
Synthesis of Compound 14:
To a solution of compound 13 (140 mg) in THF (5 ml), TBAF (0.39 ml) is added. After 24 h additional TBAF (0.2 ml) is added and the stirring is continued for 50 h. The reaction mixture is concentrated to dryness and the crude product is purified by column chromatography (silica) to afford compound 14 (95 mg).
Synthesis of Compound 16:
A mixture of compound 14 (0.16 g) and compound 15 (0.35 g, synthesized as described by Banteli et al., Helvetica Chimica Acta 83:2893-2907, 2000) is co-evaporated with toluene twice and then dried under vacuum. The mixture is dissolved in dry CH2Cl2 (10 ml) and stirred with flame dried molecular sieve (4 A) and 2,6-di-tert-Bu-pyridine (0.59 ml) for 30 min at RT. The reaction mixture is cooled to 0° C. and MeOTf (0.25 ml) is added with stirring. The reaction mixture is stirred for 4 h at RT, filtered through a bed of Celite, washed with CH2Cl2 (2×10 ml) and then transferred to a reparatory funnel. The organic layer is washed with a cold saturated solution of NaHCO3 (25 ml) and brine (25 ml), dried (Na2SO4), filtered, and concentrated to dryness. The residue is purified by column chromatography (silica) to give compound 16 (0.23 g).
Synthesis of Compound 17:
Compound 16 (0.96 mg) is dissolved in dioxane-water (10:2, 12 ml) and AcOH (0.2 ml) is added. 10% Pd/C (0.8 g) is added and stirred vigorously under hydrogen (40 psi) for 16 h at RT. The reaction mixture is filtered through a bed of Celite and washed with MeOH. Solvent is evaporated off to give compound 17 (700 mg).
Synthesis of Compound 18:
Compound 17 (500 mg) is treated at RT with 0.01N NaOMe in MeOH (20 ml) for 1 h. The reaction is neutralized with AcOH and the solvent is evaporated off to give compound 18 (300 mg).
Synthesis of Compound 19:
Compound 18 (200 mg) is dissolved in ethylenediamine (3 ml) and the solution is stirred for 3 h at 70° C. Solvent is evaporated off and the residue is purified by Sep-Pak C18 column to give compound 19 (160 mg).
Synthesis of Compound 21:
Commercially available (Aldrich Chemical Co., Milwaukee, Wi.) compound 20 (1.47 g) is suspended in CH2Cl2 (70 ml). To this suspension is added a solution of (BOC)2O (3.86 g in 70 ml of CH2Cl2) dropwise with stirring at RT. The stirring is continued for 2 h. The reaction mixture is concentrated to dryness and purified by column chromatography (CombiFlash) to give compound 21 (1.8 g).
Synthesis of Compound 23:
A suspension of compound 21 (1.59 g), commercially available (Aldrich Chemical Co., Milwaukee, Wis.) compound 22 (0.8 g) and K2CO3 (0.48 g) in DMF (15 ml) is stirred at 60° C. overnight. The reaction mixture is concentrated to a thick oil and filtered through a glass syringe filter, dissolved in CH2Cl2 and purified by column chromatography (silica) to give compound 23 (1.96 g).
Synthesis of Compound 24:
To a cold (0° C.) solution of compound 23 (0.99 g) in THF (30 ml) is added LiAlH4 (2M solution in THF, 3.05 ml) with stirring. Stirring is continued for 2 h at 0° C. The reaction is quenched with EtOAc and diethylether is added. The mixture is transferred to a reparatory funnel and washed with cold saturated solution of NH4Cl. The organic layer is dried (Na2SO4), filtered, and concentrated to dryness. The residue is purified column chromatography (CombiFlash) to give compound 24 (717 mg).
Synthesis of Compound 25:
A solution of (COCl)2 (0.15 ml) in CH2Cl2 (3 ml) is cooled down to −78° C. To this solution is added DMSO (0.25 ml) dropwise in the cold (−78° C.) with stirring and stirring is continued for 15 min at −78° C. Compound 24 (717 mg) in CH2Cl2 (3 ml) is added dropwise to the above mixture at −78° C. with stirring. The stirring is continued for 15 min at −78° C. and DIPEA (1.17 ml) is added and stirred for 15 min. The reaction mixture is warmed to RT slowly. The reaction mixture is concentrated to dryness and the crude product is purified by column chromatography (silica) to give compound 25 (701 mg).
Synthesis of Compound 26:
Compound 25 (77 mg) is dissolved in CH2Cl2 (7 ml) and CF3COOH (1.4 ml) is added with stirring. The reaction mixture is stirred at RT for 2 h, CF3COOH (0.7 ml) is added and stirring is continued for another 1 h. The reaction mixture is evaporated to dryness and purified by Sep-Pak C18 Cartridges to give compound 26 (30 mg).
Synthesis of Compound 27:
To a solution of compound 19 (5 mg) in DMSO (0.2 ml) is added compound 26 (4 mg) and NaBH3CN (0.8 mg, 0.08 ml from a stock solution of 10 mg/ml) and AcOH (0.8 mg, 0.08 ml from a stock solution of 10 mg/ml). The reaction mixture is stirred at 60° C. for 2 h and the solvent is evaporated off. The residue is purified by HPLC (reverse phase C18 column) to give compound 27 (2.5 mg) which is heterobifunctional Compound #1 (also referred to herein as “Compound #1”).
Example 2
Synthesis of Heterobifunctional Compound #2 (Compound 28 of FIG. 2)
Synthesis of Intermediate II:
(−)-Shikimic acid (20 g) in MeOH (200 ml) and sulfuric acid (2 ml, 98%) are stirred at rt for 50 h. The reaction mixture is neutralized with 2N aqueous NaOH in the cold. After evaporation to dryness, the residue is purified by silica gel chromatography to afford II (19.2 g).
Synthesis of Intermediate (III):
Methyl shikimate (II, 10 g), 2,2 dimethoxypropane (10 ml) and p-TsOH (0.8 g) are dissolved in acetonitrile (125 ml) and stirred at rt for 1 h. The reaction mixture is then neutralized with triethylamine (2 ml) and evaporated to dryness. The residue is chromatographed on silica gel to yield III (11 g).
Synthesis of Intermediate IV:
The shikimic acid derivative III (10 g) and PtO2/C (10%, 250 mg) in MeOH (40 ml) are hydrogenated at rt under vigorous stirring. After 16 h the reaction mixture is filtered over celite and evaporated to dryness. The residue is chromatographed on silica gel to yield IV.
Synthesis of Intermediate V:
To a solution of IV (8 g) in DCM (100 ml) at 0° C. are added pyridine (12 ml), acetic anhydride (7 ml) and a DMAP (25 mg). The reaction mixture is stirred at rt for 1 h, and diluted with EtOAc (250 ml). After washing with 0.5 M aqueous HCl (3×50 ml), saturated solution of KHCO3 (3×50 ml) and brine (3×50 ml), the combined organic layers are dried (Na2SO4) and evaporated to dryness. The residue is purified by chromatography on silica gel to yield V (6.8 g).
Synthesis of Intermediate VI:
A solution of V (6.0 g) in acetic acid (30 ml, 80%) is stirred at 80° C. for 1 h. Solvent is evaporated off and the residue is purified by chromatography on silica gel (DCM/MeOH 14:1) to yield VI (3.6 g).
Synthesis of Intermediate (VII):
A solution of VI (3 g) and p-TsCl (3.5 g) in pyridine (30 ml) is stirred at rt for 6 h. MeOH (5 ml) is added and the solvent is evaporated at reduced pressure, the residue dissolved in EtOAc (3×150 ml) and the organic layers are washed with 0.5 M aqueous HCl (0° C.), water (cold) and brine (cold). The combined organic layers are dried (Na2SO4), filtered on Celite and evaporated to dryness. The residue is purified by chromatography on silica gel (toluene/EtOAc 4:1) to yield VII (3.7 g).
Synthesis of Compound VIII:
A solution of VII (3 g) and NaN3 (2.5 g) in DMF (20 ml) is stirred at 80° C. The reaction mixture is cooled to rt and diluted with EtOAc (200 ml) and water (50 ml). The organic layer is additionally washed twice with water (2×50 ml) and once with brine (50 ml). All aqueous layers are extracted twice with EtOAc (2×50 ml). The combined organic layers are dried with Na2SO4, filtered and the solvent is evaporated off. The residue is purified by chromatography on silica gel (petroleum ether/EtOAc 5:2) to give VIII (2.2 g).
Synthesis of Compound X:
To a solution of ethyl 2,3,4-tri-O-benzyl-α-L-fucothiopyanoside IX (1.5 g) in DCM (3 ml), bromine (150 μl) is added at 0° C. under argon. After 5 min the cooling bath is removed and the reaction mixture is stirred for additional 25 min at rt. Cyclohexene (200 μl) is added and the reaction mixture is added to a solution of VIII (400 mg), (Et)4NBr (750 mg) and powdered 4 Å molecular sieves in DCM (10 ml) and DMF (5 ml). After 16 h, triethylamine (1.5 ml) is added and stirred for an additional for 10 min, diluted with EtOAc (50 ml) and washed with sat. aqueous NaHCO3, water and brine. The aqueous layers are extracted twice with EtOAc (2×50 ml). The combined organic layers are dried (Na2SO4), filtered and evaporated to dryness. The residue is purified by chromatography on silica gel (toluene/EtOAc 9:1) to yield X (700 mg).
Synthesis of Compound XI:
To a solution of X (1.5 g) in MeOH (20 ml) is added freshly prepared NaOMe (80 mg) and the reaction mixture is stirred in a pressure tube at 80° C. for 20 h. The reaction mixture is cooled to rt and neutralized with acetic acid. Solvent is evaporated to dryness and the residue is dissolved in ether. Freshly prepared diazomethane is added and the excess diazomethane is neutralized with acetic acid. Solvent is evaporated off to give XI (1.25 g).
Synthesis of Building Block XV:
This synthesis is done exactly in same way as described previously (Helvetica Chemica Acta 83:2893-2907 (2000)).
Synthesis of Compound XVI:
A mixture of XI (1.6 g), XV (3 g) and activated powdered molecular sieves 4 Å (1 g) in DCM (17 ml) is stirred at rt under argon for 2 h. Then DMTST (2 g) is added in 4 equal portions over a period of 1.5 h. After 24 h the reaction mixture is filtered over Celite and the filtrate is diluted with DCM (100 ml). The organic layer is washed with sat. aqueous NaHCO3 and brine and the aqueous layers are extracted twice with DCM. The combined organic layers are dried (Na2SO4), filtered and evaporated to dryness. The residue is purified by chromatography on silica gel (toluene/EtOAc 8:1) to yield XVI (1.5 g).
Synthesis of Compound XVII:
To a solution of XVI (500 mg) and orotic acid chloride (500 mg) in dichloromethane (10 ml) is added a solution of triphenylphosphine (500 mg in 5 ml dichloromethane) dropwise during 10 min. The reaction mixture is stirred at rt for 25 h and the solvent is evaporated off. The residue is purified (chromatography on silica gel DCM/MeOH 19:1) to give XVII (250 mg).
Synthesis of Compound XVIII:
To a solution of XVII (200 mg) in dioxane-water (5:1, 12 ml) is added 10% Pd—C (100 mg) and the reaction mixture is stirred vigorously under hydrogen (55 psi) for 24 h. Catalyst is filtered through a bed of celite and the solvent is evaporated off. Residue is purified by silica gel chromatography to give compound XVIII (150 mg).
Synthesis of XIX:
To a solution of compound XVIII (145 mg) in MeOH (5 ml) is added a solution of NaOMe in MeOH (25%, 0.025 ml) and the reaction mixture is stirred at rt for 4 h, neutralized with acetic acid and the solvent is evaporated off. Residue is dissolved in water and passed through a bed of Dowex 50wX-8 (Na-form) resin. Water wash is evaporated off to afford compound XIX (100 mg).
Synthesis of EDA-XIX:
XIX (80 mg) is heated at 70° C. with ethylenediamine (EDA) (1 ml) with stirring for 5 h. Solvent is evaporated off and the purified by sephadex G-25 column to give EDA-XIX (82 mg).
Synthesis of Compound 28:
Compound 26 of Example 1 is reacted with EDA-XIX (and the product purified) using the procedures described in Example 1 (for the synthesis of compound 27) to give compound 28 which is heterobifunctional Compound #2 (also referred to herein as “Compound #2”).
Example 3
Assay to Assess Binding of Compounds to CXCR4
Methods
The assay assesses the ability of glycomimetic compounds to inhibit binding of an anti-CXCR4 antibody conjugated to phycoerythrin (“PE”), to CXCR4 on the surface of SupT1 cells. SupT1 cells are a T lymphoblast derived from a lymphoblastic leukemia and constitutively express CXCR4 on the cell surface. The cells are purchased from ATCC (ATCC number CRL-1942). Anti-human CXCR4-phycoerythrin monoclonal antibody (anti-CXCR4-PE) is purchased from R&D Systems (catalog number FAB170P, clone 12G5). The cells are grown in RPMI 1640 medium supplemented with 10% FBS. Approximately 2×106 cells are washed three times by centrifuging the cells at 400×g for 10 minutes and the cell pellet is resuspended in PBS plus 0.05% BSA. After the third centrifugation, the cell pellet is resuspended in PBS plus BSA to a concentration of 5×105 cells per ml. To block non-specific binding, human 1 g is added to the cells to a concentration of 1 μg per 105 cells. Next, 200 μl (1×105 cells) are added to 5 ml polypropylene round-bottom tubes (Falcon 2063 tubes). Compound #1 (Example 1) (lot 31-190) is added to the cells at final concentrations of 0.5, 5, 10, and 50 μM. To achieve a final concentration of 0.5 μM, 2.2 μl of 50 μM Compound #1 plus 19.8 μl of PBS/BSA are added to 200 μl of cells. To achieve a final concentration of 5 μM, 22 μl of 50 μM Compound #1 are added to 200 μl of cells. To achieve a final concentration of 10 μM, 4.4 μl of 500 μM Compound #1 plus 17.6 μl of PBS/BSA are added to 200 μl of cells. To achieve a final concentration of 50 μM, 22 μl of 500 μM Compound #1 are added to 200 μl of cells. Other aliquots of cells are treated with either 1 or 5 μM of the bicyclam CXCR4 antagonist AMD-3100 (Sigma Aldrich, catalog #A5602). To achieve a final concentration of 1 μM AMD-3100, 4.4 μl of 50 μM AMD-3100 plus 17.6 μl of PBS/BSA are added to 200 μl of cells and to achieve a final concentration of 5 μM, 22 μl of 50 μM AMD-3100 are added to 200 μl of cells. In addition, one tube of cells is treated with 1 μg/ml of SDF-1α (R&D Systems catalog #350-NS), the natural ligand of CXCR4. The tubes are placed at 4° C. for 15 minutes. Subsequently, each tube receives 10 μl of anti-CXCR4-PE, except one tube of cells receives 10 μl of mouse IgG 2A isotype control antibody. The tubes are incubated at 4° C. for 45 minutes. The cells are washed twice with PBS plus 0.05% BSA and the final cell pellet is resuspended in 100 μl of PBS/BSA. To fix the samples, 100 μl of 2% formaldehyde (Polysciences, Inc. ultrapure EM grade, catalog number 04018) are added to each tube. Flow cytometry is performed using a Cytomation MoFlo instrument.
Results
As shown in the table below, Compound #1 inhibits binding of anti-CXCR4-PE to SupT1 cells in a dose-dependent manner with an IC50 of 8.25 μM (FIG. 3). SDF-1α efficiently inhibits binding of the antibody to CXCR4.
















Mean





fluorescence
Median fluorescence



% positive
intensity
intensity



















SupT1 cells only
1.13
2.80
2.55


Isotype control
1.46
4.73
2.46


No inhibitor
99.28
104.75
86.60


0.5 μM Compound #1
98.69
68.49
54.25


  5 μM Compound #1
81.48
22.25
13.34


 10 μM Compound #1
42.06
14.08
6.04


 50 μM Compound #1
12.80
11.08
3.92


  1 μM AMD-3100
57.57
15.60
7.77


  5 μM AMD-3100
12.42
9.98
3.92


  1 μg/ml SDF-1α
12.37
10.25
4.07









Example 4
E-Selectin Activity
Binding Assay
Methods
The inhibition assay to screen glycomimetic antagonists of E-selectin is a competitive binding assay, which allows the determination of IC50 values. Briefly, E-selectin/Ig chimera is immobilized by incubation at 37° C. in 96 well microtiter plates for 2 hours. To reduce nonspecific binding, bovine serum albumin is added to each well and incubated at room temperature for 2 hours. The plate is washed and serial dilutions of the test compounds are added to the wells in the presence of conjugates of biotinylated, sLea polyacrylamide with streptavidin/horseradishperoxidase and incubated for 2 hours at room temperature. To determine the amount of sLea bound to immobilized E-selectin after washing, the peroxidase substrate, 3,3′,5,5′ tetramethylbenzidin (TMB) is added. After 3 minutes, the enzyme reaction is stopped by the addition of H3PO4 and the absorbance of light at a wavelength of 450 nm is determined. The concentration of test compound required to inhibit binding by 50% is determined and reported as the IC50 value for each glycomimetic E-selectin antagonist. In addition to reporting the absolute IC50 value as measured above, relative IC50 values are determined by a ratio of the IC50 measured for the test compound to that of an internal control (reference) stated for each assay.
Results
The results for heterobifunctional Compound #1 are shown in FIGS. 4 and 5. Compound #1 is a potent E-selectin antagonist (as well as possessing anti-CXCR4 activity—FIG. 3).
All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

## Claims

1. A heterobifunctional compound for inhibition of E-selectin and the CXCR4 chemokine receptor, comprising E-selectin inhibitor-Linker-CXCR4 chemokine receptor inhibitor, or a physiologically acceptable salt thereof, where the E-selectin inhibitor is not an antibody.

2. The compound of claim 1 wherein the E-selectin inhibitor is a small organic molecule.

3. The compound of claim 1 wherein the E-selectin inhibitor consists of:




wherein:
L=end of bond to Linker;
R1=H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, OH, or NHX where X═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, or OH; C(═O)OX, alkanyl substituted with C(═O)OX, C(═O)NHX, alkanyl substituted with C(═O)NHX, where X═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, or OH; C(═O)X, OX, NHX, NHC(═O)X, where X═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, or OH;
R2=—OH,





—O—C(═O)—X, —NH2, —NH—C(═O)—NHX, or —NH—C(═O)—X where n=0-2 and X is independently selected from C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl,




where Q is H or a physiologically acceptable salt, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, aryl, (CH2)m-aryl where m is 1-10, and where n=0-10, and any of the above ring compounds may be substituted with one to three independently selected of Cl, F, CF3, C1-C8 alkoxy, NO2, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, C1-C14 aryl, or OY, C(═O)OY, NY2 or C(═O)NHY where Y is H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, or C1-C14 aryl;
R3═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, CN, CH2CN, C(═O)X where X is H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, NHOH, NHOCH3, NHCN, or NX2, or C(═O)OY where Y is H, C alkanyl, C alkenyl or C1-C8 alkynyl; and





where the cyclopropane ring may be substituted with one to two, and the cyclohexane ring may be substituted with one to three, independently selected of Cl, F, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl or OY where Y is H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl or C1-C14 aryl.

4. The compound of claim 3 wherein the E-selectin inhibitor consists of:




wherein L=end of bond to Linker.

5. The compound of claim 3 wherein the E-selectin inhibitor consists of:




wherein L=end of bond to Linker.

6. The compound of claim 3 wherein the E-selectin inhibitor consists of:




wherein L=end of bond to Linker.

7. The compound of claim 3 wherein the E-selectin inhibitor consists of:




wherein L=end of bond to Linker.

8. The compound of claim 1 or claim 2 wherein the CXCR4 chemokine receptor inhibitor consists of:




wherein L=end of bond to Linker.

9. The compound of claim 1 having the formula:




wherein:
R1═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, OH, or NHX where X═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, or OH; C(═O)OX, alkanyl substituted with C(═O)OX, C(═O)NHX, alkanyl substituted with C(═O)NHX, where X═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, or OH; C(═O)X, OX, NHX, NHC(═O)X, where X═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, halogenated C1-C8 alkanyl, aryl which may be substituted with one or more of Me, OMe, halide, or OH;
R2=—OH,





—O—C(═O)—X, —NH2, —NH—C(═O)—NHX, or —NH—C(═O)—X where n=0-2 and X is independently selected from C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl,




where Q is H or a physiologically acceptable salt, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, aryl, (CH2)m-aryl where m is 1-10, and where n=0-10, and any of the above ring compounds may be substituted with one to three independently selected of Cl, F, CF3, C1-C8 alkoxy, NO2, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, C1-C14 aryl, or OY, C(═O)OY, NY2 or C(═O)NHY where Y is H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, or C1-C14 aryl;
R3═H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, CN, CH2CN, C(═O)X where X is H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl, NHOH, NHOCH3, NHCN, or NX2, or C(═O)OY where Y is H, C1-C8 alkanyl, C1-C8 alkenyl or C1-C8 alkynyl; and





where the cyclopropane ring may be substituted with one to two, and the cyclohexane ring may be substituted with one to three, independently selected of Cl, F, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl or OY where Y is H, C1-C8 alkanyl, C1-C8 alkenyl, C1-C8 alkynyl or C1-C14 aryl.

10. The compound of claim 1 having the formula:

11. The compound of claim 1 having the formula:

12. The compound of claim 1 having the formula:

13. The compound of claim 1 having the formula:

14. The compound of any one of claims 1-7, or 9-13, where Linker is one of:
(a) —C(═O)—NH—(CH2)2—NH—;
(b) —CH2—NH—CH2—; and
(c) —C(═O)—NH—CH2—.

15. A method for inhibiting metastasis of a cancer in an individual who is in need thereof, comprising administering to the individual a compound in an amount effective for inhibiting metastasis, wherein the compound is according to claim 1 or claim 2 with or without a pharmaceutically acceptable carrier or diluent.

16. A method for inhibiting metastasis of a cancer in an individual who is in need thereof, comprising administering to the individual a compound in an amount effective for inhibiting metastasis wherein the compound is according to any one of claim 3, 9, 10 or 13 with or without a pharmaceutically acceptable carrier or diluent.

17. A method for mobilizing cancer cells from a site into the bloodstream and retaining the cancer cells in the bloodstream in an individual who is in need thereof, comprising administering to the individual a compound in an amount effective for said mobilizing and said retaining of the cancer cells, wherein the compound is according to claim 1 or claim 2 with or without a pharmaceutically acceptable carrier or diluent.

18. A method for mobilizing cancer cells from into the bloodstream and retaining the cancer cells in the bloodstream in an individual who is in need thereof, comprising administering to the individual a compound in an amount effective for said mobilizing and said retaining of the cancer cells, wherein the compound is according to any one of claim 3, 9, 10 or 13 with or without a pharmaceutically acceptable carrier or diluent.

19. A method for releasing cells into circulating blood and enhancing retention of the cells in the blood of an individual who is need of such treatment, comprising administering to the individual a compound in an amount effective for treatment, wherein the compound is according to claim 1 or claim 2 with or without a pharmaceutically acceptable carrier or diluent.

20. The method of claim 19, further including the step of collecting the cells released.

21. The method of claim 20 wherein the step of collecting utilizes apheresis.

22. The method of claim 19 wherein the cells are bone marrow progenitor cells.

23. A method for releasing cells into circulating blood and enhancing retention of the cells in the blood of an individual who is need of such treatment, comprising administering to the individual a compound in an amount effective for treatment, wherein the compound is according to any one of claim 3, 9, 10 or 13 with or without a pharmaceutically acceptable carrier or diluent.

24. A method for the treatment of an inflammatory disease in which the adhesion or migration of cells occurs in the disease in an individual who is in need of such treatment, comprising administering to the individual a compound in an amount effective for treatment, wherein the compound is according to claim 1 or claim 2 with or without a pharmaceutically acceptable carrier or diluent.

25. A method for the treatment of an inflammatory disease in which the adhesion or migration of cells occurs in the disease in an individual who is in need of such treatment, comprising administering to the individual a compound in an amount effective for treatment, wherein the compound is according to any one of claim 3, 9, 10 or 13 with or without a pharmaceutically acceptable carrier or diluent.

26. The compound of any one of claims 1-7, and 9-13 in combination with a pharmaceutically acceptable carrier or diluent.

27. The compound of claim 9 or claim 10 having the formula:




where Me is methyl.

28. The compound of claim 9 or claim 13 having the formula:




where Me is methyl.

29. The method of claim 23 wherein the cells are bone marrow progenitor cells.

30. The method of claim 23 further including the step of collecting the cells released.

31. The method of claim 30 wherein the step of collecting utilizes apheresis.

32. The compound of claim 8 where Linker is one of:
(a) —C(═O)—NH—(CH2)2—NH—;
(b) —CH2—NH—CH2—; and
(c) —C(═O)—NH—CH2—.

33. The compound of claim 8 in combination with a pharmaceutically acceptable carrier or diluent.